0001]The present disclosure, mutant nitrile hydratase, nucleic acid encoding the mutant nitrile hydratase, the expression vectors and transformants comprising the nucleic acid, the method of producing said variant nitrile hydratase, and a method for preparation of amide compounds.
BACKGROUND
[0002]Nitrile hydratase, an enzyme having a nitrile hydration activity of converting a nitrile group of various compounds into an amide group through hydration, and is utilized in the production process of industrial amide compounds using enzymatic reactions.
[0003]
　Recently, the required level for producing technical industrial amide compounds tends to have become higher and higher. In view of these circumstances, in order to reduce the running cost of the nitrile hydratase occupied in the production cost of the amide compound, various studies have been made so far. In particular, nitrile for hydratase, the technology according to the variants which can improve the activity value per unit weight of the enzyme, have been made many reports (JP-A-9-275978, JP-2004 -194588, JP 2005-160403, JP-WO 2004/056990, WO 2010/055666, JP 2007-143409, JP JP 2008-253182, etc.).
[0004]
　Here, in a typical preparation process of industrial amide compounds using enzymatic reactions, usually in solution was adjusted to a pH of 7-9, by using the catalytic action of a nitrile hydratase nitrile a reaction step of the compound the reaction is allowed to proceed for synthesizing an amide compound, the pH of the solution was adjusted to 3.5-6.5, the adsorption nitrile hydratase present in the solution using activated carbon in the pH and purified to obtain an amide compound which is purified by removing by using did in this order (Japanese Unexamined Patent Publication No. 2001-270857).
Summary of the Invention
Problems that the Invention is to Solve
[0005]
　The present inventors, the production efficiency of industrial amide compound using nitrile hydratase intensive studies in order to improve more than ever. It Consequently, even in the purification step performed in the pH range of acidic, that the nitrile compound by using the catalytic action of a nitrile hydratase continues to proceed the reaction for synthesizing an amide compound, which is effective as the design guideline It was heading.
[0006]
　However, in general, the optimum pH of the wild-type nitrile hydratase is 7-9, wild-type nitrile hydratase activity is significantly decreased in the conditions of pH 3.5 ~ 6.5. Therefore, even with wild-type nitrile hydratase, an enzyme reaction in the purification step is fully
not occur to the extent that the legs. Further, various nitrile hydratase variant as described above have been reported, it also activity is significantly decreased in acidic conditions such as those used in the purification process. Thus, technology related nitrile hydratase variant having good pH stability of the enzyme activity that catalyzes the reaction for synthesizing an amide compound from a nitrile compound in a purification step under acidic conditions, not been reported so far It was. In relatively strong acidic conditions such as in particular pH5.0 or less, protein is higher tendency to deactivate, techniques relating to such nitrile hydratase variant enzyme activity is satisfactorily maintained even under pH conditions, this It had not been reported to date.
[0007]
　Accordingly, the present disclosure, stability synthesis reaction of the amide compound from a nitrile compound to the pH of the enzyme activity that catalyzes is improved, to provide a technique relating mutant nitrile hydratase having a novel mutation point. More specifically, also provides a technique relating mutant nitrile hydratase that good retention of enzymatic activity in an acidic region such as pH 3.5 ~ 6.5. More specifically, pH provides a technique relating mutant nitrile hydratase that good retention of enzymatic activity even at relatively strong acid region of 3.5 and 5.0.
Means for Solving the Problems
[0008]
　The present disclosure includes the following aspects.
At least one amino acid residue in pseudotype Nocardia thermophila-derived nitrile hydratase, selected from the group consisting of amino acid residue substitutions of the following (a) ~ (e) having a <1> alpha-subunit and β-subunit comprising a substitution mutant nitrile hydratase,
(a) alpha substitution of the N-terminus of the subunit to the Asn of the 40 amino acid residues,
the 43 th amino acid residue from the N-terminus of (b) alpha-subunit Val substitution
of, (c) substitution of the N-terminus of the β-subunit to 205 th amino acid residue of
Val, (d) substitution of the N-terminus of the β-subunit to 206 th amino acid residue of
Gln, (e ) substitution of the N-terminus of the β-subunit to 215 th amino acid residue Asn.
[0009]
<2> comprises an amino acid residue substitution (a) 2 or more amino acid residue substitutions selected from the group consisting of ~ (e), a mutant nitrile hydratase according to <1>.
[0010]
<3> an amino acid residue substitution and (b), the amino acid residue substitutions (a), (c), at least one selected from the group consisting of (d) and (e), including <1> or <2 mutant nitrile hydratase according to>.
[0011]
In the amino acid sequence of <4> alpha-subunit, 36th amino acid from the N terminus is Trp, <1> ~ <3> mutant nitrile hydratase according to any one of.
[0012]
<5> in the amino acid sequence of the α-subunit, 36th amino acid from the N terminus is Met, Ser, Gly or Ala, <1> ~ mutant nitrile hydratase according to any one of <3>.
[0013]
<6> alpha amino acid sequence of the subunit, satisfy one or more of the following (1) to (13), <1> to <5> mutant nitrile hydratase according to any one
　of: ( 1) N 6 amino acid residue from the terminus is Thr or
　Ala, (2) amino acid residues 13 th from N-terminal is
　Leu, (3) 19 amino acid residue from the N-terminus at Val
　there, (4) amino acid residues 27 th from N-terminal is
　Ile, (5) from the N-terminal 48 amino acid residues which are
　Gln, 71 amino acid residues from (6) N-terminal His
　is, (7) 92 amino acid residue from the N-terminus is
　Glu, (8) 94 th amino acid residue from the N-terminus is
　Ile, the 126th amino acid residue from (9) N-terminal it is tyr,
　or (10) N-terminal Et 148th amino acid residue is
　Asp, (11) amino acid residues 188 th from N-terminal is
　Gly, a (12) 197 amino acid residue from the N-terminal
　Cys, (13) N 204 th amino acid residue from the terminus is Arg.
[0014]
<7> amino acid sequence of the β subunit, satisfies at least one of the following (15) to (47), <1> - mutant nitrile hydratase according to any one of
　<6>: ( 4 th amino acid residue from 15) N-terminal is
　Met, (16) 8 th amino acid residue from the N-terminus is
　Ala, a (17) 10 th amino acid residue from the N-terminal Asp,
　(18) 24th amino acid residue from the N-terminus is
　Ile, (19) 33 th amino acid residue from the N terminus is Val or
　Met, (20) N 37 th amino acid residue from the terminus Val or
　Leu, (21) 40 amino acid residue from the N-terminus is Ile, Val or
　Leu, (22) 41 amino acid residue from the N-terminus is
　Ile, 46 from (23) N-terminal Amino acid residues eye is Lys,
　(24) from the N-terminal 48 amino acid residues which are
　Val, (25) 51 th amino acid residue from the N terminus is
　Val, from (26) N-terminal 61 th amino acid residue is Val, Gly, Trp, Ser, Leu or
　Thr, (27) 79 th amino acid residue from the N terminus is
　Asn, 96 th amino acid residue from (28) N-terminal There is Arg,
　(29) 107 amino acid residue from the N-terminus is
　Met, (30) 108 amino acid residue from the N-terminus is Asp or
　Arg, the 110th amino acid residue from (31) N-terminal Asn in
　it, (32) amino acid residues 112 th from N-terminal is Val or
　Ile, (33) 118 amino acid residue from the N terminus is
　Val, 127 amino acid residues from (34) N-terminal group is
　Ser, (35) amino acid residues 146 th from N-terminal is
　Gly, (36) 0.99 amino acid residue from the N-terminus is Asn or
　Ser, 160 th from (37) N-terminal amino acid residues are Cys, Trp or
　Met, (38) 168 amino acid residue from the N-terminus is Glu,
　176 th a from (39) N-terminal Amino acid residue is Ala, Thr, Met or
　Cys, (40) 186 amino acid residue from the N-terminus is
　Arg, which is the 200th amino acid residue from (41) N-terminal
　Glu, ( 42) 212 amino acid residue from the N terminus is
　Tyr, a (43) 217 amino acid residue from the N-terminal Val, His, Met, Gly, Ser, Leu or Cys,
　(44) amino acid residues 218 th from N-terminal is Met or
　Ser, (45) 226 amino acid residue from the N-terminus is
　Ile, the 230th amino acid residue from (46) N-terminal Glu in
　it, (47) 231 amino acid residue from the N terminus is Val.
[0015]
<8> In any one of the pseudotyped Nocardia thermophila-derived nitrile hydratase following [1] to [49], comprising at least one amino acid residue substitutions selected from the group consisting of said (a) ~ (e) <1> mutant nitrile hydratase according to any one of to <7>.
　[1] and the α-subunit having the amino acid sequence of SEQ ID NO: 1, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 2,
　and α subunit having an amino acid sequence of [2] SEQ ID NO: 16 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 33,
　[3] and the α-subunit having the amino acid sequence of SEQ ID NO: 17, and β-subunit having the amino acid sequence of SEQ ID NO: 33, the nitrile hydratase having,
　[4] and α subunit having an amino acid sequence of SEQ ID NO: 18, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 34,
　the amino acid sequence of [5] SEQ ID NO: 19 Nitoriruhi having a subunit α with the β subunit having the amino acid sequence of SEQ ID NO: 34, the Dorataze,
　nitrile hydratase, having a β-subunit having α and subunit, the amino acid sequence of SEQ ID NO: 35 having an amino acid sequence of [6] SEQ ID NO: 20
　sub-α having the amino acid sequence of [7] SEQ ID NO: 20 nitrile hydratase having the unit, the β subunit having the amino acid sequence of SEQ ID NO: 36, and
　[8] and α subunit having an amino acid sequence of SEQ ID NO: 21, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 37,
　and α subunit having an amino acid sequence of [9] SEQ ID NO: 21 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 38,
　[10] and the α-subunit having the amino acid sequence of SEQ ID NO: 21, and the β subunit having the amino acid sequence of SEQ ID NO: 39, the nitrile hydratase having,
　[11] and α subunit having an amino acid sequence of SEQ ID NO: 21, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 40,
　the amino acid sequence of [12] SEQ ID NO: 18 has a subunit α with the β subunit having the amino acid sequence of SEQ ID NO: 41, the Nitrile hydratase,
　and α subunit having an amino acid sequence of [13] SEQ ID NO: 18, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 42,
　having the amino acid sequence of [14] SEQ ID NO: 21 and α-subunit, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 43,
　sub-β with α and subunit, the amino acid sequence of SEQ ID NO: 44 having an amino acid sequence of [15] SEQ ID NO: 22 nitrile hydratase has a unit, a
　nitrile hydratase having a β-subunit having the amino acid sequence of [16] and α subunit having an amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 45,
　[17] and α subunit having an amino acid sequence of SEQ ID NO: 24, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 46,
　and α subunit having an amino acid sequence of [18] SEQ ID NO: 25 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 47,
　[19] and α subunit having an amino acid sequence of SEQ ID NO: 18, and β-subunit having the amino acid sequence of SEQ ID NO: 48, the nitrile hydratase has,
　and α subunit having an amino acid sequence of [20] SEQ ID NO: 23, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 49,
　the amino acid sequence of [21] SEQ ID NO: 16 and the subunit α with the β subunit having the amino acid sequence of SEQ ID NO: 50, Yu Nitrile hydratase that,
　the α-subunit having the amino acid sequence of [22] SEQ ID NO: 26, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 51,
　the amino acid sequence of [23] SEQ ID NO: 27 and the subunit α with nitrile hydratase having a beta subunit having an amino acid sequence of SEQ ID NO: 52,
　beta has the amino acid sequence of the α and the subunit, SEQ ID NO: 53 having an amino acid sequence of [24] SEQ ID NO: 28 nitrile hydratase has a subunit, a
　[25] and α subunit having an amino acid sequence of SEQ ID NO: 17, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 54,
　[26] and α subunit having an amino acid sequence of SEQ ID NO: 29, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 55,
　and α subunit having an amino acid sequence of [27] SEQ ID NO: 18 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 56,
　[28] and α subunit having an amino acid sequence of SEQ ID NO: 18, and β-subunit having the amino acid sequence of SEQ ID NO: 57, the nitrile hydratase has,
　and α subunit having an amino acid sequence of [29] SEQ ID NO: 30, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 58,
　the amino acid sequence of [30] SEQ ID NO: 29 and the subunit α with the β subunit having the amino acid sequence of SEQ ID NO: 59, Yu Nitrile hydratase that,
　the α-subunit having the amino acid sequence of [31] SEQ ID NO: 31, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 60,
　the amino acid sequence of [32] SEQ ID NO: 18 and α subunit having nitrile hydratase having a beta subunit having an amino acid sequence of SEQ ID NO: 61,
　beta has a α-subunit having the amino acid sequence of [33] SEQ ID NO: 32, the amino acid sequence of SEQ ID NO: 62 nitrile hydratase has a subunit, a
　[34] and α subunit having an amino acid sequence of SEQ ID NO: 30, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 63,
　[35] and α subunit having an amino acid sequence of SEQ ID NO: 30, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 64,
　and α subunit having an amino acid sequence of [36] SEQ ID NO: 30 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 65,
　[37] and α subunit having an amino acid sequence of SEQ ID NO: 25, and β-subunit having the amino acid sequence of SEQ ID NO: 54, the nitrile hydratase having,
　[38] and α subunit having an amino acid sequence of SEQ ID NO: 30, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 66,
　the amino acid sequence of [39] SEQ ID NO: 1 Yusuke and the subunit α with the β subunit having the amino acid sequence of SEQ ID NO: 67, the That nitrile hydratase,
　and α subunit having an amino acid sequence of [40] SEQ ID NO: 1, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 68,
　the amino acid sequence of [41] SEQ ID NO: 1 and the subunit α with the beta subunit having an amino acid sequence of SEQ ID NO: 69, nitrile having hydratase,
　[42] and α subunit having an amino acid sequence of SEQ ID NO: 1, beta has the amino acid sequence of SEQ ID NO: 70 nitrile hydratase has a subunit, a
　[43] and α subunit having an amino acid sequence of SEQ ID NO: 1, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 71,
　[44] and α subunit having an amino acid sequence of SEQ ID NO: 21, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 72,
　and α subunit having an amino acid sequence of [45] SEQ ID NO: 1 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 73,
　[46] [1] to the N-terminus of the definitive α subunits to any one of the nitrile hydratase [45] 36 th amino acid residue was Trp residues, nitrile hydratase
　[47] above [1] ~ alpha subunits or the alpha subunit of one of the nitrile hydratase of the [46] (a) and 90% and α subunit variant consisting of an amino acid sequence having a sequence identity or more, beta subunits or the beta Sa of the nitrile hydratase (a) Unit and the β-subunit variant consisting of an amino acid sequence having 90% or more sequence identity to a nitrile hydratase having, alpha subunit and β at least one of alpha-subunit variant or β subunit of the subunit is a variant, the nitrile hydratase
　added in α-subunit in [48] above [1] one of the nitrile hydratase of the ~ [46] (B), substitutions, deletions, and / or inserted amino acid residue the total number of 1 to 10 (excluding the amino acid residues are substituted in the (a) and (b)) group, and, in the β-subunit in the nitrile hydratase (B), addition, substitution, deletion , and / or the total number 1 of the inserted amino acid residue (except the amino acid residues are substituted for the (c) ~ (e)) 10 A nitrile hydratase
[0016]
<9> <1> to nucleic acid encoding a mutant nitrile hydratase according to any one of <8>.
[0017]
Vector comprising a nucleic acid according to <10> <9>.
[0018]
<11> is an expression vector, vectors according to <10>.
[0019]
<12> transformant comprising the expression vector according to <11>.
[0020]
<13> transformants be cultured in a medium, and at least from one of the transformants and medium cultured according to <12>, according to any one of <1> to <8> method for producing a mutant nitrile hydratase comprising recovering the mutant nitrile hydratase.
[0021]
<14> mutant nitrile hydratase obtained by the production method according to <13>.
[0022]
<15> <1> to <8> and comprising contacting the mutant nitrile hydratase of a nitrile compound according to any one of <14> The production method of the amide compound.
[0023]
<16> Further, in pH 3.5 ~ pH 6.5, and removing impurities from a solution containing an amide compound, the production method of the amide compound according to <15>.
[0024]
<17> further comprising purifying the amide compound activated carbon manufacturing method of the amide compound according to <15> or <16>.
Effect of the invention
[0025]
　According to the present disclosure, stability synthesis reaction of the amide compound from a nitrile compound to the pH of the enzyme activity that catalyzes is improved, it is possible to provide a technique relating mutant nitrile hydratase having a novel mutation point. More specifically, it is possible to provide a technique relating mutant nitrile hydratase that good retention of enzymatic activity in an acidic region such as pH 3.5 ~ 6.5. More specifically, it is possible to provide a technique relating mutant nitrile hydratase pH is satisfactorily retain the enzymatic activity even at relatively strong acid region of 3.5 and 5.0.
DESCRIPTION OF THE INVENTION
[0026]

　The disclosure relates in pseudotype Nocardia thermophila-derived nitrile hydratase and a α-subunit and β-subunit, from the group consisting of amino acid residue substitutions of the following (a) ~ (e) chosen at least one amino acid residue substitution mutant nitrile hydratase (hereinafter, also referred to as mutant nitrile hydratase a) provides:
(a) from the N-terminus of α-subunit of 40 amino acid residues substitution of
Asn, (b) alpha-substituted from the N-terminus of the subunit to the 43 th amino acid residue of
Val, substitution of Val of the amino acid residues from the N-terminal 205th of (c) beta
subunit ( d) substitution of the N-terminus of the beta subunit to the 206 th amino acid residue of
Gln, 215 th a from the N-terminus of (e) beta subunit Substitution of Asn at amino acid residue.
[0027]
　Above (a) ~ mutant amino acid residue substitutions comprising at least one or more (e) has not been reported so far. That is, the amino acid residue substitutions of the including the mutant nitrile hydratase (a) ~ (e) can be regarded as either a previously reported has not been made mutations. The amino acid residue substitution of (a) ~ (e), hereinafter also referred to as amino acid residue substitution groups A. The mutant nitrile hydratase according to the present disclosure including the mutations described above, the synthesis reaction of the amide compound from a nitrile compound to the enzyme activity with stability in a wide pH range as compared with the conventional nitrile hydratase variant an enzyme capable of exhibiting. This will be described in detail in Example.
[0028]
　Those have been in an attempt to achieve a variant of a wild-type pseudotyped Nocardia thermophila-derived nitrile hydratase Conventionally, these are for the purpose of mainly improving the enzymatic activity at optimum pH conditions of the enzyme a is, the optimum pH for the enzyme put opposite acidic conditions
has not been a study for the enzyme activity that. Enzyme activity of the nitrile hydratase under acidic conditions to deviate from optimal conditions, which has been first noted by the present inventors, in which found that new amino acid residue mutations described above is effective.
[0029]
　The following describes in more detail a mutant nitrile hydratase according to the present disclosure. Description of the nucleotide sequences of the present disclosure, there is a case where a nucleic acid strand that holds the nucleotide sequence is made in consideration of the arrangement of the chains of one even when forming the double strand, in the other strand of the duplex, the description of such a sequence should be applied replaced to its complementary sequence.
　The term "step" in the present disclosure, not only separate steps, the intended purpose of the process even if that can not be clearly distinguished from other processes if it is achieved, are included in this term.
　Numerical range expressed by using "to" in the present disclosure represents a range including the respective minimum and maximum values of the numerical values described before and after "to".
　In the present disclosure, the amount of each component in the composition, if substances corresponding to the component in the composition there are a plurality, unless otherwise specified, means the total amount of the plural substances present in the composition to.
[0030]
　In the present disclosure, the pseudotyped Nocardia thermophila-derived nitrile hydratase, of wild type having the β subunit having the amino acid sequence of the α-subunit SEQ ID NO: 2 having the amino acid sequence of SEQ ID NO: 1 pseudotyped Nocardia thermophila Philadelphia-derived nitrile hydratase as well, relative to a wild-type shoe de Nocardia thermophila-derived nitrile hydratase, to the extent that one of ordinary skill in the art can be recognized as a modified sequence of the wild-type shoe de Nocardia thermophila-derived nitrile hydratase is a concept including a modified nitrile hydratase plus modified. Modifications nitrile hydratase is included in the concept of pseudotyped Nocardia thermophila-derived nitrile hydratase, relative to a wild-type pseudotyped Nocardia thermophila-derived nitrile hydratase, another in (i) 1 or more amino acid residues the substitution of an amino acid residue, (ii) above (a) ~ (e) 1 or more amino acid residues deleted except, (iii) insertion of amino acid residues, the amino acid sequence of (iv) alpha-subunit N-terminal and the addition of amino acid residues to either or both of the C-terminal, (v) the addition of amino acid residues to one or both of the N-terminal and C-terminal amino acid sequence of the β subunit, the group consisting of modified nitrile hydratase plus one or more modifications selected from is also included.
[0031]
　In α subunit of the modified nitrile hydratase, the number of amino acid residues substituted is, for example, 1 to 20, or 1 to 15 or 1 to 10, or 1 to 8, or a 1-7 or a 1-6, 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 be zero.
　In β-subunit of the modified nitrile hydratase, the number of amino acid residues substituted is, for example, 1 to 20, or 1 to 15 or 1 to 10, or 1 to 8, or a 1-7 or a 1-6, 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 be zero.
[0032]
　In α subunit of the modified nitrile hydratase, the number of deleted amino acid residues, for example, 1 to 10, or a 1-7 or a 1-4, or be 1-2 , or a 1. The number of deleted amino acid residues may be zero.
　In β-subunit of the modified nitrile hydratase, the number of deleted amino acid residues, for example, 1 to 10, or a 1-7 or a 1-4, or be 1-2 , or a 1. The number of deleted amino acid residues may be zero.
[0033]
　In α subunit of the modified nitrile hydratase, the number of inserted amino acid residue, for example, 1 to 10, or a 1-7 or a 1-4, or a 1-2, or a 1. The number of inserted amino acid residue may be zero.
　In β-subunit of the modified nitrile hydratase, the number of inserted amino acid residue, for example, 1 to 10, or a 1-7 or a 1-4, or a 1-2, or a 1. The number of inserted amino acid residue may be zero.
[0034]
　In α subunit of the modified nitrile hydratase, substitutions, the total number of deletions or inserted amino acid residue, for example, from 1 to 20, or 1 to 14 or 1 to 8, or 1 ~ is 4, or 1 to 2, or 1. Such as when there is a terminal additions, substitutions, the total number of deletions or inserted amino acid residue may be zero.
　In β-subunit of the modified nitrile hydratase, substitutions, the total number of deletions or inserted amino acid residue, for example, from 1 to 20, or 1 to 14 or 1 to 8, or 1 ~ is 4, or 1 to 2, or 1. Such as when there is a terminal additions, substitutions, the total number of deletions or inserted amino acid residue may be zero.
[0035]
　In α subunit of the modified nitrile hydratase, the number of terminal added amino acid residue is, for example, 1 to 60 per terminal, or 1-40, or 1 to 20 or 1 to it is 10, or 1 to 5 or 1 to 3 or 1. The number of added amino acid residue may be zero.
　In β-subunit of the modified nitrile hydratase, the number of terminal added amino acid residue is, for example, 1 to 60 per terminal, or 1-40, or 1 to 20 or 1 to it is 10, or 1 to 5 or 1 to 3 or 1. The number of added amino acid residue may be zero.
　Endcapping amino acid residue may be, for example, a secretory signal sequence. Terminal additional amino acids residues only the N-terminus, only the C-terminal, or may be present in both the N-terminal and C-terminal.
[0036]
　It said modified nitrile hydratase The similarity with the wild-type pseudotyped Nocardia thermophila-derived nitrile hydratase can be represented by sequence identity.
　The amino acid sequence of the α subunit of the modified nitrile hydratase, between the amino acid sequence of the α-subunit of the wild type pseudotyped Nocardia thermophila-derived nitrile hydratase of SEQ ID NO: 1, for example 70% or more sequence identity, alternatively from 80% or more sequence identity, alternatively from 85% or more sequence identity, or have 90% or more sequence identity, or 95% or more sequence identity It has a sex, or have 96% or more sequence identity, alternatively from 97% or more sequence identity, alternatively from 98% or more sequence identity, or 99% sequence identity to a.
[0037]
　The amino acid sequence of the β subunit of the modified nitrile hydratase, between the amino acid sequence of the β-subunit of the wild type pseudotyped Nocardia thermophila-derived nitrile hydratase of SEQ ID NO: 2, for example, more than 70% sequence identity, alternatively from 80% or more sequence identity, alternatively from 85% or more sequence identity, or have 90% or more sequence identity, or 95% or more sequence identity It has a sex, or have 96% or more sequence identity, alternatively from 97% or more sequence identity, alternatively from 98% or more sequence identity, or 99% sequence identity to a.
[0038]
　Incidentally, alignment of sequences each other, can be done in ClustalW (1.83), initial parameters (gap open penalty: 10, Gap extension Pena
can be done using: Luthi containing 0.05).
[0039]
　If the amino acid sequence of the modified nitrile hydratase was amino acid sequence alignment of wild-type shoe de Nocardia thermophila-derived nitrile hydratase, depending on the style of the modification of the modified nitrile hydratase, the amino acid residues with each other to cope with on top of the alignment even, it is the distance from the N-terminus of subunit defined as above are different. In the present disclosure, such a case, the modified nitrile hydratase, the X amino acid residue from the N-terminus of the subunit, the N-terminus of the subunit of the wild type pseudotyped Nocardia thermophila-derived nitrile hydratase from points to the X amino acid residue and alignment on the corresponding amino acid residues. For example, "40 th amino acid residue from the N-terminus of the alpha subunit" in the present disclosure, "alpha 40 th Asp residues from the N-terminus of subunit" or "40 th amino acid residues from the N-terminus of the alpha subunit the Asp residue "is a group, in the amino acid sequence of the modified nitrile hydratase may be be located from the N-terminus of α-subunit in positions other than 40 th. For example, 41 amino acid residue from the N-terminus of the α subunit of the modified nitrile hydratase (not necessarily an Asp) is a result of the insertion of an amino acid residue, a wild-type pseudotyped Nocardia thermophila-derived nitrile hydratase may may correspond from the N-terminus of α-subunit of the synthetase to Asp residues is 40 amino acid residues. In this case, the N-terminus of the "alpha 40 th amino acid residue from the N-terminus of subunit", "40 th Asp residues from the N-terminus of alpha subunit" or "alpha subunit in the description in this disclosure from the Asp residue "is a 40 amino acid residue, refers to the 41 amino acid residues from the N-terminus of the α subunit in the modified nitrile hydratase.
[0040]
　In wild-type pseudotyped Nocardia thermophila-derived nitrile hydratase, the Met residues α subunits located at the beginning of SEQ ID NO: 1 from the N-terminal amino acid sequence first is an amino acid residue, the α-subunit 40 th amino acid residue from the N-terminal amino acid sequence is Asp residue, 43 amino acid residue from the N-terminus of the amino acid sequence of the α-subunit is Ala residues. In the wild-type pseudotyped Nocardia thermophila-derived nitrile hydratase, Met residues located at the beginning of SEQ ID NO: 2 is one amino acid residue from the N-terminal amino acid sequence of subunit beta, beta sub amino acid residues from the N-terminal 205 th amino acid sequence of the units is Gly residues, beta amino acid residue from the N-terminal 206 th amino acid sequence of the subunit is Pro residue, the amino acid sequence of the beta subunit amino acid residues from the N-terminal 215-th is Tyr residue.
[0041]
　Amino acid residue substitutions (a) ~ 1 or more sequence of the modified nitrile hydratase can be used as an introduction target of (e) is registered in the GenBank National Center for Biotechnology Information which (NCBI) offered sequence of the nitrile hydratase, or choose from the nitrile hydratase of the sequences described in known documents may be used. Further, JP-A-9-275978 discloses the above, JP 2004-194588, JP 2005-160403, JP-WO 2004/056990, modified nitrile as described in WO 2010/055666 choose from hydratase of the array can be used. In this case, in addition to the improvements described in these patent documents, it is possible to obtain new effects of improving the pH stability by one or more introduction of amino acid residue substitutions (a) ~ (e).
[0042]
　Incidentally, the modified nitrile hydratase can be used as one or more introduction subject of the amino acid residue substitutions (a) ~ (e), the 36th amino acid residue from the N-terminus (wild-type pseudotyped Nocardia thermophila (in the following, the amino acid residue substitutions (f) filler amino acid residue corresponding on alignments 36th Thr residue from the N-terminal in the amino acid sequence from nitrile hydratase) is those wherein Trp residues also called) met it may be. Having the amino acid residue substitutions (a) ~ amino acid residue substitutions in addition to one or more of (e) (f) is preferable from the viewpoint of obtaining a mutant nitrile hydratase that has a more improved pH stability. Saying
In other had, when the amino acid residue substitutions (f) is present, in stronger tends effect amino acid residue substitutions from (a) by (e).
[0043]
　Further, for example, WO 2010/055666, to the amino acid sequence of the wild-type pseudotyped Nocardia thermophila-derived nitrile hydratase, the following amino acid residue substitutions (hereinafter, an amino acid residue substitution groups B It has been described to introduce also referred) and.
[0044]
　alpha (represented by position relative to the N-terminus of the alpha subunit) amino acid residue substitutions in the amino acid sequence of subunit
　6 th Leu, an amino acid residue substituted with Thr or Ala
　Ile is 13 amino acid residues substituted Leu to
　which is a 19 amino acid residue Ala substituted with Val
　replacing Met which is 27 amino acid residues Ile
　substituting Thr is 36 amino acid residues Met, Ser, the Gly or Ala
　48 th Asn, the amino acid residues Gln substitution
　of Arg which is 71 amino acid residues substituted with His
　and Asp is the 92nd amino acid residue substituted with Glu
　to Met is the 94th amino acid residues Ile substituted
　replace a 126th amino acid residue Phe to Tyr
　place the Gly is a 148 amino acid residues Asp
　188 th Thr, the amino acid residues Gly substitution
　to Gly is a 197 amino acid residue substituted with Cys
　substituted for Val is a 204 amino acid residues Arg
[0045]
　beta (represented by position relative to the N-terminus of the beta-subunit) amino acid residue substitutions in the amino acid sequence of subunit
　fourth the Val is an amino acid residue substitution with Met
　is 8 amino acid residues Gly and Ala substituted
　10 amino acid residues in which Thr substitution at Asp
　which is the 24th amino acid residue Val substituted Ile
　is 33 amino acid residues Ala to Val or Met substitution
　at 37th amino acid residue substituted some Phe Val or Leu
　and Thr is 40 amino acid residues Ile, Val or substituted Leu
　is 41 amino acid residues Phe substitution at Ile
　to Met is the 46th amino acid residues Lys substitution
　of a 48 amino acid residue Leu substituted with Val
　replace Phe is 51 amino acid residues Val
　6 -Th Ala is an amino acid residue Val, Gly, Trp, Ser, Leu or substituted Thr
　His-a 79 amino acid residue substituted with Asn
　replace a 96 amino acid residue Gln to Arg
　107 th which is the amino acid residue Pro substituted with Met
　replace Glu is the 108th amino acid residues Asp or Arg
　110 th Glu, an amino acid residue substitution in Asn
　112 amino acid residues in which the Lys Val or Ile substitution
　118th amino acid residue in a Phe Val to replace
　a 127th amino acid residue Leu substituted with Ser to
　replace Arg which is 146 amino acid residues Gly
　Ala Asn or substituted with Ser is 150 amino acid residues
　replace Arg is 160 amino acid residues Cys, with Trp or Met
　168 amino acid residue is a Thr substitution at Glu
　Ala Tyr, which is 176 amino acid residues, Thr, Met or substituted with Cys
　substituted for Leu is a 186 amino acid residues Arg
　200 amino acid residues in it the Ala substituted with Glu
　replacing Pro is a 206 amino acid residues Leu
　2 The second amino acid residue is a Ser substitution at Tyr
　Val to Asp is 217 amino acid residues, His, Met, Gly, Ser , Leu or substituted Cys
　Cys a Met or a 218 amino acid residue substituted with Ser
　substituting Val is a 226 amino acid residues Ile
　replace a 230th amino acid residue Ala to Glu
　Replacing Ala is a 231 amino acid residues Val
[0046]
　Modified nitrile hydratase can be used as one or more introduction subject of the amino acid residue substitutions (a) ~ (e) when compared to wild-type pseudotyped Nocardia thermophila-derived nitrile hydratase amino acid sequence in may have may be one containing one or more amino acid residue substitutions selected from the amino acid residue substitution groups B, further amino acid residue substitutions to (f). The amino acid residue substitutions may be included in a combination of two or more. Examples of such combinations are described many also in WO 2010/055666. Further, modified nitrile hydratase, of course, should have a nitrile hydratase activity.
[0047]
　For example, if it contains a combination of three,
　a combination of Ala33Val in Thr36Ser and Asp92Glu and β subunit in α-subunit,
　the Ala61Gly and Ala150Asn in Met94Ile and β subunit in α-subunit combination,
　Val4Met in β-subunit the combination of Tyr176Ala and Asp217Val
　Ala33Met in β-subunit, a combination of His79Asn and Tyr176Thr,
　Thr40Val in β-subunit, a combination of Cys218Met and Val226Ile,
　and the like as examples.
[0048]
　Further, for example if it contains a combination of eight is,
　Ile13Leu in α-subunit, Ala19Val, Arg71His and Phe126Tyr and Phe37Leu in β-subunit, Gln96Arg, combinations of Glu108Asp and Ala200Glu (WO 2010/055666 Transformation body No. 59),
　Leu6Thr in α-subunit, Met27Ile, Thr10Asp in Thr36Met and Phe126Tyr and β subunit, Pro107Met, combinations of Phe118Val and Ala200Glu (WO 2010/055666: transformant No. 68),
　in the α-subunit Leu6Thr, Thr36Met and Phe126Tyr And Thr10Asp in β-subunit, Phe118Val, Ala200Glu, Pro206Leu and combinations Ala230Glu (WO 2010/055666: transformant No. 92),
　Leu6Thr in α-subunit, Ala19Val and Phe126Tyr parallel
Leu48Val in β-subunit in beauty, His79Asn , Glu108Arg, Ser212Tyr and combinations Ala230Glu (WO 2010/055666: transformant No. 85),
　Thr36Met in α-subunit, Gly148Asp and Val204Arg and Phe41Ile in β-subunit, Phe51Val, Glu108Asp, Pro206Leu and Ala230 The combination of lu (WO 2010/055666: transformant number 93),
　There are mentioned as examples.
[0049]
　Also, one or more amino acid sequence of the modified nitrile hydratase can be used as the introduction target, wild-type pseudotyped Nocardia thermophila-derived nitrile hydratase amino of the amino acid residue substitutions (a) ~ (e) compared to sequences may contain amino acid residue substitution at amino acid residue other than the illustrated position (amino acid residue substitution group B and amino acid residue substitutions (f)) above. Such one or more amino acid residue substitutions and / or amino acid residues in addition to the substitution (f), or substitutions of amino acid residues of the amino acid residues of the amino acid residue substitution groups B of the amino acid residue substitutions group B without substitution (f), nor to modification nitrile hydratase comprising an amino acid residue substitution at amino acid residue other than amino acid residue substitution groups B and amino acid residue substitutions (f), the amino acid residue substitutions (a ) one or more introduction of ~ (e), through stabilization of the conformational as described below, the effect of improving the pH stability with the modified nitrile hydratase.
[0050]
　With respect to the wild-type pseudotyped Nocardia thermophila-derived nitrile hydratase or modified nitrile hydratase, by introducing one or more of the amino acid residue substitutions (a) ~ (e), variants of the present disclosure it is possible to obtain a nitrile hydratase.
[0051]
　The mutant nitrile hydratase, of the amino acid residue substitutions (a) ~ (e) and (f), may include two combinations of amino acid residue substitutions shown in Table I.
[0052]
[Table 1]

[0053]
　The mutant nitrile hydratase, of the amino acid residue substitutions (a) ~ (e) and (f), may contain also three combinations of amino acid residue substitutions shown in Table II.
[0054]
[Table 2]

[0055]
　The mutant nitrile hydratase, of the amino acid residue substitutions (a) ~ (e) and (f), may include four combinations of amino acid residue substitutions shown in Table III.
[0056]
[table 3]

[0057]
　The mutant nitrile hydratase, of the amino acid residue substitutions (a) ~ (e) and (f), may include five combinations of amino acid residue substitutions shown in Table IV.
[0058]
[Table 4]

[0059]
　The mutant nitrile hydratase may comprise a combination of amino acid residue substitutions (a) ~ (e) and (f) of all (six amino acid residue substitutions shown in Table V).
[0060]
[table 5]

[0061]
　Amino acid residue substitutions (a) 1 or more amino acid sequence of the modified nitrile hydratase can be used as an introduction target of ~ (e), the modified nitrile hydratase produced by WO 2010/055666, etc. it may be amino acid sequences or sequences similar thereto. Therefore, mutant nitrile hydratase according to the present disclosure, for example,
　alpha substitution of the N-terminus of the subunit to the Asn of the 40 th amino acid residue,
　from the N-terminus of the alpha subunit of 43 amino acid residues Val substitution of,
　replacement of the N-terminus of the β-subunit to 205 th amino acid residue of Val,
　substitution of the N-terminus of the β-subunit to 206 th amino acid residue of Gln,
　from the N-terminus of β-subunit 215 substitution of Asn of the amino acid residues.
　At least one of those introduced in any of the following nitrile hydratase (1) - (47) (hereinafter also referred to as mutant nitrile hydratase B) may be:
[0062]
　(1) and the α-subunit having the amino acid sequence of SEQ ID NO: 1, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 2,
　and α subunit having the amino acid sequence of (2) SEQ ID NO: 16 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 33,
　(3) and the α-subunit having the amino acid sequence of SEQ ID NO: 17, and β-subunit having the amino acid sequence of SEQ ID NO: 33, the nitrile hydratase having,
　(4) and α-subunit having the amino acid sequence of SEQ ID NO: 18, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 34,
　the amino acid sequence of (5) SEQ ID NO: 19 and the subunit α with the β subunit having the amino acid sequence of SEQ ID NO: 34, Yu Nitrile hydratase that,
　the α-subunit having the amino acid sequence of (6) SEQ ID NO: 20, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 35,
　the amino acid sequence of (7) SEQ ID NO: 20 and the subunit α with the beta subunit having an amino acid sequence of SEQ ID NO: 36, nitrile having hydratase,
　beta has a subunit α having the amino acid sequence of (8) SEQ ID NO: 21, the amino acid sequence of SEQ ID NO: 37 nitrile hydratase has a subunit, a
　(9) and α subunit having an amino acid sequence of SEQ ID NO: 21, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 38,
　(10) and the α-subunit having the amino acid sequence of SEQ ID NO: 21, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 39,
　and α subunit having the amino acid sequence of (11) SEQ ID NO: 21 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 40,
　(12) and the α-subunit having the amino acid sequence of SEQ ID NO: 18, and β-subunit having the amino acid sequence of SEQ ID NO: 41, the nitrile hydratase has,
　(13) and the α-subunit having the amino acid sequence of SEQ ID NO: 18, amino of SEQ ID NO: 42
nitrile hydratase having a β-subunit having an acid sequence,
　(14) amino acids of SEQ ID NO: 21 and α subunit having the sequence, beta Sabuyu having the amino acid sequence of SEQ ID NO: 43 Tsu DOO and, nitrile hydratase having,
　(15) and α-subunit having the amino acid sequence of SEQ ID NO: 22, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 44,
　(16) SEQ ID NO: and α subunit having an amino acid sequence of the 23, of SEQ ID NO: 45 and the subunit β having the amino acid sequence, the nitrile hydratase having,
　(17) and the α-subunit having the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 46 nitrile hydratase having a β-subunit having the amino acid sequence, and
　(18) and the α-subunit having the amino acid sequence of SEQ ID NO: 25, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 47,
　(19) and the α-subunit having the amino acid sequence of SEQ ID NO: 18, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 48,
　and α subunit having the amino acid sequence of (20) SEQ ID NO: 23 , a β subunit having the amino acid sequence of SEQ ID NO: 49, nitrile having hydratase,
　(21) and the α-subunit having the amino acid sequence of SEQ ID NO: 16, and β-subunit having the amino acid sequence of SEQ ID NO: 50, the nitrile hydratase has,
　the α-subunit having the amino acid sequence of (22) SEQ ID NO: 26, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 51,
　the amino acid sequence of (23) SEQ ID NO: 27 and the subunit α with, beta Sabuyu having the amino acid sequence of SEQ ID NO: 52 Tsu DOO and, nitrile hydratase having,
　(24) and α-subunit having the amino acid sequence of SEQ ID NO: 28, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 53,
　(25) SEQ ID NO: and the subunit α with the amino acid sequence of 17, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 54,
　(26) and α-subunit having the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 55 nitrile hydratase having a β-subunit having the amino acid sequence, and
　(27) and the subunit α with the amino acid sequence of SEQ ID NO: 18, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 56,
　(28) and the α-subunit having the amino acid sequence of SEQ ID NO: 18, and β-subunit having the amino acid sequence of SEQ ID NO: 57, nitrile having hydratase,
　and α subunit having the amino acid sequence of (29) SEQ ID NO: 30 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 58,
　and the subunit α having the amino acid sequence of (30) SEQ ID NO: 29, and β-subunit having the amino acid sequence of SEQ ID NO: 59, the nitrile hydratase has,
　the α-subunit having the amino acid sequence of (31) SEQ ID NO: 31, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 60,
　the amino acid sequence of (32) SEQ ID NO: 18 and the subunit α with, beta Sabuyu having the amino acid sequence of SEQ ID NO: 61 Tsu DOO and, nitrile hydratase having,
　(33) and α-subunit having the amino acid sequence of SEQ ID NO: 32, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 62,
　(34) SEQ ID NO: and the subunit α with the amino acid sequence of 30, and β-subunit having the amino acid sequence of SEQ ID NO: 63, nitrile having hydratase,
　(35) and α-subunit having the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 64 nitrile hydratase having a β-subunit having the amino acid sequence, and
　(36) and the α-subunit having the amino acid sequence of SEQ ID NO: 30, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 65,
　(37) and α-subunit having the amino acid sequence of SEQ ID NO: 25, and β-subunit having the amino acid sequence of SEQ ID NO: 54, nitrile having hydratase,
　and α subunit having the amino acid sequence of (38) SEQ ID NO: 30 , a β subunit having the amino acid sequence of SEQ ID NO: 66, nitrile having hydratase,
　(39) and α-subunit having the amino acid sequence of SEQ ID NO: 1, and the β subunit having the amino acid sequence of SEQ ID NO: 67, the nitrile hydratase has,
　the α-subunit having the amino acid sequence of (40) SEQ ID NO: 1, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 68,
　the amino acid sequence of (41) SEQ ID NO: 1 and the subunit α with, beta subunit having an amino acid sequence of SEQ ID NO: 69 , Nitrile hydratase having,
　(42) and the α-subunit having the amino acid sequence of SEQ ID NO: 1, and the β subunit having the amino acid sequence of SEQ ID NO: 70, the having nitrile hydratase,
　(43) of SEQ ID NO: 1 amino acid and α subunit having the sequence, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 71,
　and the subunit α having the amino acid sequence of (44) SEQ ID NO: 21, the amino acid sequence of SEQ ID NO: 72 nitrile hydratase has a subunit β having, a,
　(45) and α-subunit having the amino acid sequence of SEQ ID NO: 1, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 73,
　(46) In (1) to the 36th amino acid residue from the N-terminus of the definitive α subunits to any one of the nitrile hydratase of (45) was Trp residues, nitrile
　hydratase, (47) the (1) - (46) nitrile hydratase any which is one specific nitrile hydratase α subunit amino acid sequence or α sub consisting of the amino acid sequence and amino acid sequence having at least 70% sequence identity with one of the a unit variants, a modified nitrile hydratase having a β-subunit variant consisting of an amino acid sequence having the amino acid sequence or the amino acid sequence 70% or more sequence identity with β-subunit of the specific nitrile hydratase, at least one of the amino acid sequence of the amino acid sequence and β subunits of α-subunit α is the amino acid sequence of the subunit variants or β-subunit variant, modified nitrile hydratase.
[0063]
　The above nitrile hydratase (2) to (45), a nitrile hydratase having the β subunit having the amino acid sequence of the α-subunit SEQ ID NO: 2 having the amino acid sequence of SEQ ID NO: 1 and (1) Comparison then are different from the amino acid residues described in tables 6 to 13 below, it has been described in conjunction with the transformant number WO 2010/055666. In the following table, the numbers in the column of mutation site represents the position of the N-terminal amino acid sequence of subunit applicable. Further, the transformant number is a number that has been assigned in WO 2010/055666.

claims

In pseudotype Nocardia thermophila-derived nitrile hydratase and a α-subunit and β-subunit comprises at least one amino acid residue substitutions selected from the group consisting of amino acid residue substitutions of the following (a) ~ (e) , mutant nitrile hydratase,
(a) alpha substitution of the N-terminus of the subunit to the Asn of the 40 th amino acid residue,
substitution of the 43 th amino acid residue from the N-terminus of (b) alpha-subunit Val ,
substitution of the N-terminus of subunit (c) beta to 205 th amino acid residue of
Val, (d) substitution of the N-terminus of the beta subunit to the 206 th amino acid residue of
Gln, (e) beta sub substitution of the N-terminus of the unit to the 215 th amino acid residue Asn.
[Requested item 2]
　Amino acid residue substitutions (a) ~ containing two or more amino acid residue substitutions selected from the group consisting of (e), a mutant nitrile hydratase according to claim 1.
[Requested item 3]
　Amino acid residue substituted with (b), the amino acid residue substitutions (a), (c), (d) and at least one selected from the group consisting of (e), including, in claim 1 or claim 2 mutant nitrile hydratase described.
[Requested item 4]
　In the amino acid sequence of the α-subunit, 36th amino acid from the N terminus is Trp, mutant nitrile hydratase according to any one of claims 1 to 3.
[Requested item 5]
　In the amino acid sequence of the α-subunit, 36th amino acid from the N terminus is Met, Ser, Gly or Ala, the mutant nitrile hydratase according to any one of claims 1 to 3.
[Requested item 6]
　α amino acid sequence of the subunit, satisfy one or more of the following (1) to (13), according to claim 1 mutant nitrile hydratase according to any one of claims 5:
　(1) N 6 th amino acid residues from the terminus is Thr or
　Ala, (2) 13 th amino acid residue from the N terminus is
　Leu, (3) 19 amino acid residue from the N terminus is
　Val, ( 4) 27 th amino acid residue from the N-terminus is
　Ile, (5) from the N-terminal 48 amino acid residues which are
　Gln, a (6) 71 th amino acid residue from the N-terminal His,
　(7) 92 amino acid residue from the N-terminus is
　Glu, (8) 94 th amino acid residue from the N-terminus is
　Ile, is a Tyr 126 amino acid residues from (9) N-terminal ,
　from (10) N-terminal 48 th amino acid residue is
　Asp, (11) amino acid residues 188 th from N-terminal is
　Gly, a (12) 197 amino acid residue from the N-terminal
　Cys, (13) N-terminal 204 amino acid residues from is Arg.
[Requested item 7]
　β amino acid sequence of the subunit, satisfies at least one of the following (15) to (47), according to claim 1 any one mutant nitrile hydratase according to claim 6:
　(15) N 4 th amino acid residue from the terminus is
　Met, (16) amino acid residues 8 th from N-terminal is
　Ala, (17) 10 th amino acid residue from the N terminus is
　Asp, (18) 24 amino acid residue from the N-terminus is
　Ile, (19) amino acid residues 33 th from N-terminal is Val or
　Met, (20) 37 amino acid residue from the N-terminus at Val or Leu
　there, (21) amino acid residues 40 th from N-terminal is Ile, Val or
　Leu, (22) amino acid residues 41 th from N-terminal is
　Ile, (23) 46 from No. N-terminal Amino acid residues are Lys,
　(24) from the N-terminal 48 amino acid residues which are
　Val, (25) amino acid residues 51 th from N-terminal is
　Val, 61 from (26) N-terminal amino acid residue is Val, Gly, Trp, Ser, is Leu or
　Thr, (27) 79 th amino acid residue from the N terminus is
　Asn, 96 th amino acid residue from (28) N-terminal is Arg,
　(29) 107 amino acid residue from the N-terminus is
　Met, (30) 108 amino acid residue from the N-terminus is Asp or
　Arg, the 110th amino acid residue from (31) N-terminal Asn in
　it, (32) amino acid residues 112 th from N-terminal is Val or
　Ile, (33) 118 amino acid residue from the N terminus is
　Val, 127 amino acid residues from (34) N-terminal group is
　Ser, (35) amino acid residues 146 th from N-terminal is
　Gly, (36) 0.99 amino acid residue from the N-terminus is Asn or
　Ser, 160 th from (37) N-terminal amino acid residues are Cys, Trp or
　Met, (38) 168 amino acid residue from the N-terminus is Glu,
　176 th a from (39) N-terminal Amino acid residue is Ala, Thr, Met or
　Cys, (40) 186 amino acid residue from the N-terminus is
　Arg, which is the 200th amino acid residue from (41) N-terminal
　Glu, ( 42) 212 amino acid residue from the N terminus is
　Tyr, a (43) 217 amino acid residue from the N-terminal Val, His, Met, Gly, Ser, Leu or Cys,
　(44) amino acid residues 218 th from N-terminal is Met or
　Ser, (45) 226 amino acid residue from the N-terminus is
　Ile, the 230th amino acid residue from (46) N-terminal Glu in
　it, (47) 231 amino acid residue from the N terminus is Val.
[Requested item 8]
　In any of pseudotyped Nocardia thermophila-derived nitrile hydratase following [1] to [49], comprising at least one amino acid residue substitutions selected from the group consisting of said (a) ~ (e), claims mutant nitrile hydratase according to any one of 1 to claim 7.
　[1] and the α-subunit having the amino acid sequence of SEQ ID NO: 1, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 2,
　and α subunit having an amino acid sequence of [2] SEQ ID NO: 16 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 33,
　[3] and the α-subunit having the amino acid sequence of SEQ ID NO: 17, and β-subunit having the amino acid sequence of SEQ ID NO: 33, the nitrile hydratase having,
　[4] and α subunit having an amino acid sequence of SEQ ID NO: 18, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 34,
　the amino acid sequence of [5] SEQ ID NO: 19 Nitoriruhi having a subunit α with the β subunit having the amino acid sequence of SEQ ID NO: 34, the Dorataze,
　nitrile hydratase, having a β-subunit having α and subunit, the amino acid sequence of SEQ ID NO: 35 having an amino acid sequence of [6] SEQ ID NO: 20
　sub-α having the amino acid sequence of [7] SEQ ID NO: 20 nitrile hydratase having the unit, the β subunit having the amino acid sequence of SEQ ID NO: 36, and
　[8] and α subunit having an amino acid sequence of SEQ ID NO: 21, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 37,
　and α subunit having an amino acid sequence of [9] SEQ ID NO: 21 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 38,
　[10] and the α-subunit having the amino acid sequence of SEQ ID NO: 21, and the β subunit having the amino acid sequence of SEQ ID NO: 39, the nitrile hydratase having,
　[11] and α subunit having an amino acid sequence of SEQ ID NO: 21, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 40,
　the amino acid sequence of [12] SEQ ID NO: 18 has a subunit α with the β subunit having the amino acid sequence of SEQ ID NO: 41, the Nitrile hydratase,
　[13] and the α-subunit having the amino acid sequence of SEQ ID NO: 18, amino of SEQ ID NO: 42
nitrile hydratase having a β-subunit having an acid sequence,
　[14] the amino acid sequence of SEQ ID NO: 21 and the subunit α with nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 43,
　has a subunit α having the amino acid sequence of [15] SEQ ID NO: 22, the amino acid sequence of SEQ ID NO: 44 nitrile hydratase having β and the subunit, a
　nitrile hydratase having a β-subunit having the amino acid sequence of [16] and α subunit having an amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 45,
　[17] and α subunit having an amino acid sequence of SEQ ID NO: 24, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 46,
　and α subunit having an amino acid sequence of [18] SEQ ID NO: 25 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 47,
　[19] and α subunit having an amino acid sequence of SEQ ID NO: 18, and β-subunit having the amino acid sequence of SEQ ID NO: 48, the nitrile hydratase has,
　and α subunit having an amino acid sequence of [20] SEQ ID NO: 23, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 49,
　the amino acid sequence of [21] SEQ ID NO: 16 and the subunit α with the β subunit having the amino acid sequence of SEQ ID NO: 50, Yu Nitrile hydratase that,
　the α-subunit having the amino acid sequence of [22] SEQ ID NO: 26, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 51,
　the amino acid sequence of [23] SEQ ID NO: 27 and the subunit α with nitrile hydratase having a beta subunit having an amino acid sequence of SEQ ID NO: 52,
　beta has the amino acid sequence of the α and the subunit, SEQ ID NO: 53 having an amino acid sequence of [24] SEQ ID NO: 28 nitrile hydratase has a subunit, a
　[25] and α subunit having an amino acid sequence of SEQ ID NO: 17, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 54,
　[26] and α subunit having an amino acid sequence of SEQ ID NO: 29, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 55,
　and α subunit having an amino acid sequence of [27] SEQ ID NO: 18 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 56,
　[28] and α subunit having an amino acid sequence of SEQ ID NO: 18, and β-subunit having the amino acid sequence of SEQ ID NO: 57, the nitrile hydratase has,
　and α subunit having an amino acid sequence of [29] SEQ ID NO: 30, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 58,
　the amino acid sequence of [30] SEQ ID NO: 29 and the subunit α with the β subunit having the amino acid sequence of SEQ ID NO: 59, Yu Nitrile hydratase that,
　the α-subunit having the amino acid sequence of [31] SEQ ID NO: 31, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 60,
　the amino acid sequence of [32] SEQ ID NO: 18 and α subunit having nitrile hydratase having a beta subunit having an amino acid sequence of SEQ ID NO: 61,
　beta has a α-subunit having the amino acid sequence of [33] SEQ ID NO: 32, the amino acid sequence of SEQ ID NO: 62 nitrile hydratase has a subunit, a
　[34] and α subunit having an amino acid sequence of SEQ ID NO: 30, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 63,
　[35] and α subunit having an amino acid sequence of SEQ ID NO: 30, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 64,
　and α subunit having an amino acid sequence of [36] SEQ ID NO: 30 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 65,
　[37] and α subunit having an amino acid sequence of SEQ ID NO: 25, and β-subunit having the amino acid sequence of SEQ ID NO: 54, the nitrile hydratase having,
　[38] and α subunit having an amino acid sequence of SEQ ID NO: 30, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 66,
　the amino acid sequence of [39] SEQ ID NO: 1 Yusuke and the subunit α with the β subunit having the amino acid sequence of SEQ ID NO: 67, the That nitrile hydratase,
　and α subunit having an amino acid sequence of [40] SEQ ID NO: 1, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 68,
　the amino acid sequence of [41] SEQ ID NO: 1 and the subunit α with the beta subunit having an amino acid sequence of SEQ ID NO: 69, nitrile having hydratase,
　[42] and α subunit having an amino acid sequence of SEQ ID NO: 1, beta has the amino acid sequence of SEQ ID NO: 70 nitrile hydratase has a subunit, a
　[43] and α subunit having an amino acid sequence of SEQ ID NO: 1, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 71,
　[44] and α subunit having an amino acid sequence of SEQ ID NO: 21, nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 72,
　and α subunit having an amino acid sequence of [45] SEQ ID NO: 1 , nitrile hydratase having a β-subunit having the amino acid sequence of SEQ ID NO: 73,
　[46] [1] to the N-terminus of the definitive α subunits to any one of the nitrile hydratase [45] 36 th amino acid residue was Trp residues, nitrile hydratase
　[47] above [1] ~ alpha subunits or the alpha subunit of one of the nitrile hydratase of the [46] (a) and 90% and α subunit variant consisting of an amino acid sequence having a sequence identity or more, beta subunits or the beta Sa of the nitrile hydratase (a) Unit and the β-subunit variant consisting of an amino acid sequence having 90% or more sequence identity to a nitrile hydratase having, alpha subunit and β at least one of alpha-subunit variant or β subunit of the subunit is a variant, the nitrile hydratase
　added in α-subunit in [48] above [1] one of the nitrile hydratase of the ~ [46] (B), substitutions, deletions, and / or inserted amino acid residue the total number of 1 to 10 (excluding the amino acid residues are substituted in the (a) and (b)) group, and, in the β-subunit in the nitrile hydratase (B), addition, substitution, deletion , and / or the total number 1 of the inserted amino acid residue (except the amino acid residues are substituted for the (c) ~ (e)) 10 A nitrile hydratase
[Requested item 9]
　Nucleic acid encoding a mutant nitrile hydratase according to any one of claims 1 to 8.
[Requested item 10]
　Vector comprising a nucleic acid according to claim 9.
[Requested item 11]
　Is an expression vector, vector of claim 10.
[Requested item 12]
　Transformant containing the expression vector of claim 11.
[Requested item 13]
　Culturing in a medium a transformant according to claim 12, as well as from at least one of the cultured transformant and the medium, the mutant nitrile according to any one of claims 1 to 8 method for producing a mutant nitrile hydratase comprising recovering hydratase.
[Requested item 14]
　Mutant nitrile hydratase obtained by the production method according to claim 13.
[Requested item 15]
　Comprising contacting the mutant nitrile hydratase of a nitrile compound as claimed in any one of claims 1 to 8 and claim 14, the production method of the amide compound.
[Requested item 16]
　Furthermore, in pH 3.5 ~ pH 6.5, and removing impurities from a solution containing an amide compound, the production method of the amide compound according to claim 15.
[Requested item 17]
　Moreover, an amide compound comprising purifying the activated carbon manufacturing method of the amide compound according to claim 15 or claim 16.