Nitrile hydratase variant

Abstract

A nitrile hydratase variant of the present invention comprises substitution of at least one amino acid with another amino acid to improve two or more properties of nitrile hydratase by substitution of one amino acid.

1 . A nitrile hydratase variant comprising substitution of at least one amino acid with another amino acid to improve two or more properties of nitrile hydratase by substitution of 1, 2 or 3 amino acids,
 wherein said properties to be improved are the initial reaction rate and thermal stability, and   wherein the nitrile hydratase variant comprises an α-subunit defined in SEQ ID No: 1 in the Sequence Listing and a β-subunit defined in SEQ ID No: 2 in the Sequence Listing, and substitution of at least one amino acid with another amino acid selected from substitution sites of the amino acid consisting of the following (b) to (l):   (a) 92nd of α-subunit;   (b) 94th of α-subunit;   (c) 197th of α-subunit;   (d) 4th of β-subunit;   (e) 24th of β-subunit;   (f) 79th of β-subunit;   (g) 96th of β-subunit;   (h) 107th of β-subunit;   (i) 226th of β-subunit;   (j) 110th of β-subunit and 231st of β-subunit;   (k) 206th of β-subunit and 230th of β-subunit; and   (l) 13th of α-subunit, 27th of α-subunit and 110th of β-subunit,   
       wherein
 Ile is substituted by Leu when 13th amino acid of the α-subunit is substituted, 
 Met is substituted by Ile when the 27th amino acid of the α-subunit is substituted, 
 Asp is substituted by Glu when the 92nd amino acid of the α-subunit is substituted, 
 Met is substituted by Ile when the 94th amino acid of the α-subunit is substituted, 
 Gly is substituted by Cys when the 197th amino acid of the α-subunit is substituted, 
 Val is substituted by Met when the 4th amino acid of the β-subunit is substituted, 
 Val is substituted by Ile when the 24th amino acid of the β-subunit is substituted, 
 His is substituted by Asn when the 79th amino acid of the β-subunit is substituted, 
 Gln is substituted by Arg when the 96th amino acid of the β-subunit is substituted, 
 Pro is substituted by Met when the 107th amino acid of the β-subunit is substituted, 
 Glu is substituted by Asn when the 110th amino acid of the β-subunit is substituted, 
 Pro is substituted by Leu when the 206th amino acid of the β-subunit is substituted, 
 Val is substituted by Ile when the 226th amino acid of the β-subunit is substituted, 
 Ala is substituted by Glu when the 230th amino acid of the β-subunit is substituted, and 
 Ala is substituted by Val when the 231st amino acid of the β-subunit is substituted. 
 
     
     
         2 . The nitrile hydratase variant according to  claim 1 , comprising substitution of at least one amino acid with another amino acid selected from substitution sites of the amino acid consisting of the following (m) to (x):
 (m) in case of (b) or (g), 13th Ile in the α-subunit is substituted by Leu;   (n) in case of (b) or (h), 27th Met in the α-subunit is substituted by Ile;   (o) (d) and (f);   (p) in case of (f), 230th Ala in the β-subunit is substituted by Glu;   (q) (a) and (i);   (r) in case of (i), 13th Ile in the α-subunit is substituted by Leu and 206th Pro in the β-subunit is substituted by Leu;   (s) in case of (a) and (d), 206th Pro in the β-subunit is substituted by Leu;   (t) in case of (c) and (h), 230th Ala in the β-subunit is substituted by Glu;   (u) in case of (f), 230th Ala in the β-subunit is substituted by Glu and 231st Ala in the β-subunit is substituted by Val;   (v) (a) and (e) and (i);   (w) in case of (c) and (e) and (h), 230th Ala in the β-subunit is substituted by Glu; and   (x) in case of (e) and (f), 230th Ala in the β-subunit is substituted by Glu and 231st Ala in the β-subunit is substituted by Val.   
     
     
         3 . The nitrile hydratase variant according to  claim 1 , further comprising substitution of at least one amino acid with another amino acid selected from the group consisting of (a), (c), (f), (i), (h), 230th of the β-subunit and 231st of the β-subunit in case of (e) is substituted with another amino acid. 
     
     
         4 . The nitrile hydratase variant according to  claim 1 , further comprising substitution of at least one amino acid selected from substitutions of the amino acid consisting of the following (aa) to (br):
 (aa) 36th Thr in the α-subunit is substituted by Met and 126th Phe in the α-subunit is substituted by Tyr;   (ab) 148th Gly in the α-subunit is substituted by Asp and 204th Val in the α-subunit is substituted by Arg;   (ac) 51st Phe in the β-subunit is substituted by Val and 108th Glu in the β-subunit is substituted by Asp;   (ad) 118th Phe in the β-subunit is substituted by Val and 200th Ala in the β-subunit is substituted by Glu;   (ae) 160th Arg in the β-subunit is substituted by Trp and 186th Leu in the β-subunit is substituted by Arg;   (af) 6th Leu in the α-subunit is substituted by Thr, 36th Thr in the α-subunit is substituted by Met, and 126th Phe in the α-subunit is substituted by Tyr;   (ag) 19th Ala in the α-subunit is substituted by Val, 71st Arg in the α-subunit is substituted by His, and 126th Phe in the α-subunit is substituted by Tyr;   (ah) 36th Thr in the α-subunit is substituted by Met, 148th Gly in the α-subunit is substituted by Asp, and 204th Val in the α-subunit is substituted by Arg;   (ai) 10th Thr in the β-subunit is substituted by Asp, 118th Phe in the β-subunit is substituted by Val, and 200th Ala in the β-subunit is substituted by Glu;   (aj) 37th Phe in the β-subunit is substituted by Leu, 108th Glu in the β-subunit is substituted by Asp, and 200th Ala in the β-subunit is substituted by Glu;   (ak) 37th Phe in the β-subunit is substituted by Val, 108th Glu in the β-subunit is substituted by Asp, and 200th Ala in the β-subunit is substituted by Glu;   (al) 41st Phe in the β-subunit is substituted by Ile, 51st Phe in the β-subunit is substituted by Val, and 108th Glu in the β-subunit is substituted by Asp;   (am) 46th Met in the β-subunit is substituted by Lys, 108th Glu in the β-subunit is substituted by Arg, and 212th Ser in the β-subunit is substituted by Tyr;   (an) 48th Leu in the β-subunit is substituted by Val, 108th Glu in the β-subunit is substituted by Arg, and 212th Ser in the β-subunit is substituted by Tyr;   (ao) 127th Leu in the β-subunit is substituted by Ser, 160th Arg in the β-subunit is substituted by Trp, and 186th Leu in the β-subunit is substituted by Arg;   (ap) 6th Leu in the α-subunit is substituted by Thr, 19th Ala in the α-subunit is substituted by Val, 126th Phe in the α-subunit is substituted by Tyr, 46th Met in the β-subunit is substituted by Lys, 108th Glu in the β-subunit is substituted by Arg, and 212th Ser in the β-subunit is substituted by Tyr;   (aq) 6th Leu in the α-subunit is substituted by Thr, 19th Ala in the α-subunit is substituted by Val, 126th Phe in the α-subunit is substituted by Tyr, 48th Leu in the β-subunit is substituted by Val, 108th Glu in the β-subunit is substituted by Arg, and 212th Ser in the β-subunit is substituted by Tyr;   (ar) 6th Leu in the α-subunit is substituted by Ala, 19th Ala in the α-subunit is substituted by Val, 126th Phe in the α-subunit is substituted by Tyr, 127th Leu in the β-subunit is substituted by Ser, 160th Arg in the β-subunit is substituted by Trp, and 186th Leu in the β-subunit is substituted by Arg;   (as) 6th Leu in the α-subunit is substituted by Thr, 36th Thr in the α-subunit is substituted by Met, 126th Phe in the α-subunit is substituted by Tyr, 10th Thr in the β-subunit is substituted by Asp, 118th Phe in the β-subunit is substituted by Val, and 200th Ala in the β-subunit is substituted by Glu;   (at) 19th Ala in the α-subunit is substituted by Val, 71st Arg in the α-subunit is substituted by His, 126th Phe in the α-subunit is substituted by Tyr, 37th Phe in the β-subunit is substituted by Leu, 108th Glu in the β-subunit is substituted by Asp, and 200th Ala in the β-subunit is substituted by Glu;   (au) 19th Ala in the α-subunit is substituted by Val, 71st Arg in the α-subunit is substituted by His, 126th Phe in the α-subunit is substituted by Tyr, 37th Phe in the β-subunit is substituted by Val, 108th Glu in the β-subunit is substituted by Asp, and 200th Ala in the β-subunit is substituted by Glu;   (av) 36th Thr in the α-subunit is substituted by Met, 148th Gly in the α-subunit is substituted by Asp, 204th Val in the α-subunit is substituted by Arg, 41st Phe in the β-subunit is substituted by Ile, 51st Phe in the β-subunit is substituted by Val, and 108th Glu in the β-subunit is substituted by Asp;   (aw) 148th Gly in the α-subunit is substituted by Asp, 204th Val in the α-subunit is substituted by Arg, 108th Glu in the β-subunit is substituted by Asp, and 200th Ala in the β-subunit is substituted by Glu;   (ax) 36th Thr in the α-subunit is substituted by Gly and 188th Thr in the α-subunit is substituted by Gly;   (ay) 36th Thr in the α-subunit is substituted by Ala and 48th Asn in the α-subunit is substituted by Gln;   (az) 48th Asn in the α-subunit is substituted by Glu and 146th Arg in the β-subunit is substituted by Gly;   (ba) 36th Thr in the α-subunit is substituted by Trp and 176th Tyr in the β-subunit is substituted by Cys;   (bb) 176th Tyr in the β-subunit is substituted by Met and 217th Asp in the β-subunit is substituted by Gly;   (bc) 36th Thr in the α-subunit is substituted by Ser, and 33rd Ala in the β-subunit is substituted by Val;   (bd) 176th Tyr in the β-subunit is substituted by Ala and 217th Asp in the β-subunit is substituted by Val;   (be) 40th Thr in the β-subunit is substituted by Val and 218th Cys in the β-subunit is substituted by Met;   (bf) 33rd Ala in the β-subunit is substituted by Met and 176th Tyr in the β-subunit is substituted by Thr;   (bg) 40th Thr in the β-subunit is substituted by Leu and 217th Asp in the β-subunit is substituted by Leu;   (bh) 40th Thr in the β-subunit is substituted by Ile and 61st Ala in the β-subunit is substituted by Val;   (bi) 61st Ala in the β-subunit is substituted by Thr and 218th Cys in the β-subunit is substituted by Ser;   (bj) 112th Lys in the β-subunit is substituted by Val and 217th Asp in the β-subunit is substituted by Met;   (bk) 61st Ala in the β-subunit is substituted by Trp and 217th Asp in the β-subunit is substituted by His;   (bl) 61st Ala in the β-subunit is substituted by Leu and 112th Lys in the β-subunit is substituted by Ile;   (bm) 146th Arg in the β-subunit is substituted by Gly and 217th Asp in the β-subunit is substituted by Ser;   (bn) 171st Lys in the β-subunit is substituted by Ala and 217th Asp in the β-subunit is substituted by Thr;   (bo) 150th Ala in the β-subunit is substituted by Ser and 217th Asp in the β-subunit is substituted by Cys;   (bp) 61st Ala in the β-subunit is substituted by Gly and 150th Ala in the β-subunit is substituted by Asn;   (bq) 61st Ala in the β-subunit is substituted by Ser and 160th Arg in the β-subunit is substituted by Met; and   (br) 160th Arg in the β-subunit is substituted by Cys and 168th Thr in the β-subunit is substituted by Glu.   
     
     
         5 . The nitrile hydratase variant according to  claim 1 , comprising substitutions at the following substitution sites (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII) or (XIV) with another amino acids:
 (I) 36th of α-subunit, 92nd of α-subunit, 148th of α-subunit, 204th of α-subunit, 41st of β-subunit, 51st of β-subunit and 108th of β-subunit;   (II) 6th of α-subunit, 19th of α-subunit, 94th of α-subunit, 126th of α-subunit, 46th of β-subunit, 108th of β-subunit and 212th of β-subunit;   (III) 6th of α-subunit, 19th of α-subunit, 126th of α-subunit, 4th of β-subunit, 127th of β-subunit, 160th of β-subunit and 186th of β-subunit;   (IV) 19th of α-subunit, 71st of α-subunit, 126th of α-subunit, 37th of β-subunit, 79th of β-subunit, 108th of β-subunit and 200th of β-subunit;   (V) 6th of α-subunit, 19th of α-subunit, 126th of α-subunit, 48th of β-subunit, 96th of β-subunit, 108th of β-subunit and 212th of β-subunit;   (VI) 19th of α-subunit, 71st of α-subunit, 126th of α-subunit, 37th of β-subunit, 107th of β-subunit, 108th of β-subunit and 200th of β-subunit;   (VII) 13th of α-subunit, 19th of α-subunit, 71st of α-subunit, 126th of α-subunit, 37th of β-subunit, 96th of β-subunit, 108th of β-subunit and 200th of β-subunit;   (VIII) 6th of α-subunit, 27th of α-subunit, 36th of α-subunit, 126th of α-subunit, 10th of β-subunit, 107th of β-subunit, 118th of β-subunit and 200th of β-subunit;   (IX) 6th of α-subunit, 19th of α-subunit, 126th of α-subunit, 48th of β-subunit, 79th of β-subunit, 108th of β-subunit, 212th of β-subunit and 230th of β-subunit;   (X) 6th of α-subunit, 36th of α-subunit, 126th of α-subunit, 10th of β-subunit, 118th of β-subunit, 200th of β-subunit, 206th of β-subunit and 230th of β-subunit;   (XI) 36th of α-subunit, 148th of α-subunit, 204th of α-subunit, 41st of β-subunit, 51st of β-subunit, 108th of β-subunit, 206th of β-subunit and 230th of β-subunit;   (XII) 6th of α-subunit, 19th of α-subunit, 126th of α-subunit, 48th of β-subunit, 79th of β-subunit, 108th of β-subunit, 212th of β-subunit, 230th of β-subunit and 231st of β-subunit;   (XIII) 6th of α-subunit, 19th of α-subunit, 126th of α-subunit, 46th of β-subunit, 79th of β-subunit, 108th of β-subunit, 212th of β-subunit, 230th of β-subunit and 231st of β-subunit;   (XIV) 6th of α-subunit, 19th of α-subunit, 126th of α-subunit, 24th of β-subunit, 48th of β-subunit, 79th of β-subunit, 108th of β-subunit, 212th of β-subunit, 230th of β-subunit and 231st of β-subunit.   
     
     
         6 . The nitrile hydratase variant according to  claim 5 , wherein:
 in case of (I), 36th of α-subunit is substituted with Met, 92nd of α-subunit is substituted with Glu, 148th of α-subunit is substituted with Asp, 204th of α-subunit is substituted with Arg, 41st of β-subunit is substituted with Ile, 51st of β-subunit is substituted with Val, and 108th of β-subunit is substituted with Asp;   in case of (II), 6th of α-subunit is substituted with Thr, 19th of α-subunit is substituted with Val, 94th of α-subunit is substituted with Ile, 126th of α-subunit is substituted with Tyr, 46th of β-subunit is substituted with Lys, 108th of β-subunit is substituted with Arg, and 212th of β-subunit is substituted with Tyr;   in case of (III), 6th of α-subunit is substituted with Ala, 19th of α-subunit is substituted with Val, 126th of α-subunit is substituted with Tyr, 4th of β-subunit is substituted with Met, 127th of β-subunit is substituted with Ser, 160th of β-subunit is substituted with Trp, and 186th of β-subunit is substituted with Arg;   in case of (IV), 19th of α-subunit is substituted with Val, 71st of α-subunit is substituted with His, 126th of α-subunit is substituted with Tyr, 37th of β-subunit is substituted with Val, 79th of β-subunit is substituted with Asn, 108th of β-subunit is substituted with Asp, and 200th of β-subunit is substituted with Glu;   in case of (V), 6th of α-subunit is substituted with Thr, 19th of α-subunit is substituted with Val, 126th of α-subunit is substituted with Tyr, 48th of β-subunit is substituted with Val, 96th of β-subunit is substituted with Arg, 108th of β-subunit is substituted with Arg, and 212th of β-subunit is substituted with Tyr;   in case of (VI), 19th of α-subunit is substituted with Val, 71st of α-subunit is substituted with His, 126th of α-subunit is substituted with Tyr, 37th of β-subunit is substituted with Val, 107th of β-subunit is substituted with Met, 108th of β-subunit is substituted with Asp, and 200th of β-subunit is substituted with Glu;   in case of (VII), 13th of α-subunit is substituted with Leu, 19th of α-subunit is substituted with Val, 71st of α-subunit is substituted with His, 126th of α-subunit is substituted with Tyr, 37th of β-subunit is substituted with Leu, 96th of β-subunit is substituted with Arg, 108th of β-subunit is substituted with Asp, and 200th of β-subunit is substituted with Glu;   in case of (VIII), 6th of α-subunit is substituted with Thr, 27th of α-subunit is substituted with Ile, 36th of α-subunit is substituted with Met, 126th of α-subunit is substituted with Tyr, 10th of β-subunit is substituted with Asp, 107th of β-subunit is substituted with Met, 118th of β-subunit is substituted with Val, and 200th of β-subunit is substituted with Glu;   in case of (IX), 6th of α-subunit is substituted with Thr, 19th of α-subunit is substituted with Val, 126th of α-subunit is substituted with Tyr, 48th of β-subunit is substituted with Val, 79th of β-subunit is substituted with Asn, 108th of β-subunit is substituted with Arg, 212th of β-subunit is substituted with Tyr, and 230th of β-subunit is substituted with Glu;   in case of (X), 6th of α-subunit is substituted with Thr, 36th of α-subunit is substituted with Met, 126th of α-subunit is substituted with Tyr, 10th of β-subunit is substituted with Asp, 118th of β-subunit is substituted with Val, 200th of β-subunit is substituted with Glu, 206th of β-subunit is substituted with Leu, and 230th of β-subunit is substituted with Glu;   in case of (XI), 36th of α-subunit is substituted with Met, 148th of α-subunit is substituted with Asp, 204th of α-subunit is substituted with Arg, 41st of β-subunit is substituted with Ile, 51st of β-subunit is substituted with Val, 108th of β-subunit is substituted with Asp, 206th of β-subunit is substituted with Leu, and 230th of β-subunit is substituted with Glu;   in case of (XII), 6th of α-subunit is substituted with Thr, 19th of α-subunit is substituted with Val, 126th of α-subunit is substituted with Tyr, 48th of β-subunit is substituted with Val, 79th of β-subunit is substituted with Asn, 108th of β-subunit is substituted with Arg, 212th of β-subunit is substituted with Tyr, 230th of β-subunit is substituted with Glu, and 231st of β-subunit is substituted with Val;   in case of (XIII), 6th of α-subunit is substituted with Thr, 19th of α-subunit is substituted with Val, 126th of α-subunit is substituted with Tyr, 46th of β-subunit is substituted with Lys, 79th of β-subunit is substituted with Asn, 108th of β-subunit is substituted with Arg, 212th of β-subunit is substituted with Tyr, 230th of β-subunit is substituted with Glu, and 231st of β-subunit is substituted with Val;   in case of (XIV), 6th of α-subunit is substituted with Thr, 19th of α-subunit is substituted with Val, 126th of α-subunit is substituted with Tyr, 24th of β-subunit is substituted with Ile, 48th of β-subunit is substituted with Val, 79th of β-subunit is substituted with Asn, 108th of β-subunit is substituted with Arg, 212th of β-subunit is substituted with Tyr, 230th of β-subunit is substituted with Glu, and 231st of β-subunit is substituted with Val.   
     
     
         7 . A gene encoding the nitrile hydratase variant according to  claim 1 . 
     
     
         8 . A gene encoding a nitrile hydratase variant having a gene encoding the α-subunit defined in SEQ ID No: 3 in the Sequence Listing and a gene encoding the β-subunit defined in SEQ ID No: 4 in the Sequence Listing, comprising substitution of at least one base selected from substitution sites of the base consisting of the following (b) to (l):
 (a) 274th to 276th of the base sequence of SEQ ID No: 3; 
 (b) 280th to 282nd of the base sequence of SEQ ID No: 3; 
 (c) 589th to 591st of the base sequence of SEQ ID No: 3; 
 (d) 10th to 12th of the base sequence of SEQ ID No: 4; 
 (e) 70th to 72st of the base sequence of SEQ ID No: 4; 
 (f) 235th to 237th of the base sequence of SEQ ID No: 4; 
 (g) 286th to 288th of the base sequence of SEQ ID No: 4; 
 (h) 319th to 321st of the base sequence of SEQ ID No: 4; 
 (i) 676th to 678th of the base sequence of SEQ ID No: 4; 
 (j) 328th to 330th of the base sequence of SEQ ID No: 4 and 691st to 693rd of the base sequence of SEQ ID No: 4; 
 (k) 616th to 618th of the base sequence of SEQ ID No: 4, and 688th to 690th of the base sequence of SEQ ID No: 4; and 
 (l) 37th to 39th of the base sequence of SEQ ID No: 3, 79th to 81st of the base sequence of SEQ ID No: 3, and 328th to 330th of the base sequence of SEQ ID No: 4, 
 wherein ATC is substituted by CTC when 37th to 39th of the base sequence of SEQ ID No: 3 are substituted by another base, 
 ATG is substituted by ATC when 79th to 81th of the base sequence of SEQ ID No: 3 are substituted by another base, 
 GAC is substituted by GAG when 274th to 276th of the base sequence of SEQ ID No: 3 are substituted by another base, 
 ATG is substituted by ATC when 280th to 282th of the base sequence of SEQ ID No: 3 are substituted by another base, 
 GGC is substituted by TGC when 589th to 591th of the base sequence of SEQ ID No: 3 are substituted by another base, 
 GTG is substituted by ATG when 10th to 12th of the base sequence of SEQ ID No: 4 are substituted by another base, 
 GTC is substituted by ATC when 70th to 72st of the base sequence of SEQ ID No: 4 are substituted by another base, 
 CAC is substituted by AAC when 235th to 237th of the base sequence of SEQ ID No: 4 are substituted by another base, 
 CAG is substituted by CGT when 286th to 288th of the base sequence of SEQ ID No: 4 are substituted by another base, 
 CCC is substituted by ATG when 319th to 321st of the base sequence of SEQ ID No: 4 are substituted by another base, 
 GAG is substituted by AAC when 328th to 330th of the base sequence of SEQ ID No: 4 are substituted by another base, 
 CCG is substituted by CTG when 616th to 618th of the base sequence of SEQ ID No: 4 are substituted by another base, 
 GTC is substituted by ATC when 676th to 678th of the base sequence of SEQ ID No: 4 are substituted by another base, 
 GCG is substituted by GAG when 688th to 690th of the base sequence of SEQ ID No: 4 are substituted by another base, and 
 GCC is substituted by GTC when 691th to 693th of the base sequence of SEQ ID No: 4 are substituted by another base. 
 
     
     
         9 . The gene encoding a nitrile hydratase variant according to  claim 8 , further comprising substitution of at least one base selected from substitution sites of the base consisting of the following (m) to (x):
 (m) in case of (b) or (g), 37th to 39th ATC of the base sequence of SEQ ID No: 3 are substituted by CTC;   (n) in case of (b) or (h), 79th to 81st ATG of the base sequence of SEQ ID No: 3 are substituted by ATC;   (o) (d) and (f);   (p) in case of (f), 688th to 690th GCG of the base sequence of SEQ ID No: 4 are substituted by GAG;   (q) (a) and (i);   (r) in case of (i), 37th to 39th ATC of the base sequence of SEQ ID No: 3 are substituted by CTC and 616th to 618th CCG of the base sequence of SEQ ID No: 4 are substituted by CTG;   (s) in case of (a) and (d), 616th to 618th CCG of the base sequence of SEQ ID No: 4 are substituted by CTG;   (t) in case of (c) and (h), 688th to 690th GCG of the base sequence of SEQ ID No: 4 are substituted by GAG;   (u) in case of (f), 688th to 690th GCG of the base sequence of SEQ ID No: 4 are substituted by GAG and 691st to 693rd GCC of the base sequence of SEQ ID No: 4 are substituted by GTC;   (v) (a) and (e) and (i);   (w) in case of (c) and (e) and (h), 688th to 690th GCG of the base sequence of SEQ ID No: 4 are substituted by GAG; and   (x) in case of (e) and (f), 688th to 690th GCG of the base sequence of SEQ ID No: 4 are substituted by GAG and 691st to 693rd GCC of the base sequence of SEQ ID No: 4 are substituted by GTC.   
     
     
         10 . The gene encoding a nitrile hydratase variant according to  claim 8 , further comprising substitution of at least one base with another base selected from substitution sites of the base consisting of (a), (c), (f), (i), (h), 688th to 690th of the base sequence of SEQ ID No: 4, and 691st to 693rd of the base sequence of SEQ ID No: 4, in case of (e), are substituted with another base. 
     
     
         11 . The gene encoding a nitrile hydratase variant according to  claim 8 , comprising substitution of at least one base selected from substitution sites of the base consisting of the following (aa) to (br):
 (aa) 106th to 108th ACG of the base sequence of SEQ ID No: 3 are substituted by ATG, and 376th to 378th TTC of the base sequence of SEQ ID No: 3 are substituted by TAC;   (ab) 442nd to 444th GGC of the base sequence of SEQ ID No: 3 are substituted by GAC, and 610th to 612th GTC of the base sequence of SEQ ID No: 3 are substituted by CGC;   (ac) 151st to 153rd TTC of the base sequence of SEQ ID No: 4 are substituted by GTC, and 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are substituted by GAT;   (ad) 352nd to 354th TTC of the base sequence of SEQ ID No: 4 are substituted by GTC, and 598th to 600th GCC of the base sequence of SEQ ID No: 4 are substituted by GAG;   (ae) 478th to 480th CGG of the base sequence of SEQ ID No: 4 are substituted by TGG, and 556th to 558th CTG of the base sequence of SEQ ID No: 4 are substituted by CGG;   (af) 16th to 18th CTG of the base sequence of SEQ ID No: 3 are substituted by ACG, 106th to 108th ACG of the base sequence of SEQ ID No: 3 are substituted by ATG, and 376th to 378th TTC of the base sequence of SEQ ID No: 3 are substituted by TAC;   (ag) 55th to 57th GCG of the base sequence of SEQ ID No: 3 are substituted by GTG, 211th to 213th CGT of the base sequence of SEQ ID No: 3 are substituted by CAT, and 376th to 378th TTC of the base sequence of SEQ ID No: 3 are substituted by TAC;   (ah) 106th to 108th ACG of the base sequence of SEQ ID No: 3 are substituted by ATG, 442nd to 444th GGC of the base sequence of SEQ ID No: 3 are substituted by GAC, and 610th to 612th GTC of the base sequence of SEQ ID No: 3 are substituted by CGC;   (ai) 28th to 30th ACC of the base sequence of SEQ ID No: 4 are substituted by GAC, 352nd to 354th TTC of the base sequence of SEQ ID No: 4 are substituted by GTC, and 598th to 600th GCC of the base sequence of SEQ ID No: 4 are substituted by GAG;   (aj) 109th to 111th TTC of the base sequence of SEQ ID No: 4 are substituted by CTC, 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are substituted by GAT, and 598th to 600th GCC of the base sequence of SEQ ID No: 4 are substituted by GAG;   (ak) 109th to 111th TTC of the base sequence of SEQ ID No: 4 are substituted by GTC, 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are substituted by GAT, and 598th to 600th GCC of the base sequence of SEQ ID No: 4 are substituted by GAG;   (al) 121st to 123rd TTC of the base sequence of SEQ ID No: 4 are substituted by ATC, 151st to 153rd TTC of the base sequence of SEQ ID No: 4 are substituted by GTC, and 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are substituted by GAT;   (am) 136th to 138th ATG of the base sequence of SEQ ID No: 4 are substituted by AAG, 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are substituted by CGG, and 634th to 636th TCC of the base sequence of SEQ ID No: 4 are substituted by TAC;   (an) 142nd to 144th CTG of the base sequence of SEQ ID No: 4 are substituted by GTG, 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are substituted by CGG, and 634th to 636th TCC of the base sequence of SEQ ID No: 4 are substituted by TAC;   (ao) 379th to 381st CTG of the base sequence of SEQ ID No: 4 are substituted by TCG, 478th to 480th CGG of the base sequence of SEQ ID No: 4 are substituted by TGG, and 556th to 558th CTG of the base sequence of SEQ ID No: 4 are substituted by CGG;   (ap) 16th to 18th CTG of the base sequence of SEQ ID No: 3 are substituted by ACG, 55th to 57th GCG of the base sequence of SEQ ID No: 3 are substituted by GTG, 376th to 378th TTC of the base sequence of SEQ ID No: 3 are substituted by TAC, 136th to 138th ATG of the base sequence of SEQ ID No: 4 are substituted by AAG, 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are substituted by CGG, and 634th to 636th TCC of the base sequence of SEQ ID No: 4 are substituted by TAC;   (aq) 16th to 18th CTG of the base sequence of SEQ ID No: 3 are substituted by ACG, 55th to 57th GCG of the base sequence of SEQ ID No: 3 are substituted by GTG, 376th to 378th TTC of the base sequence of SEQ ID No: 3 are substituted by TAC, 142nd to 144th CTG of the base sequence of SEQ ID No: 4 are substituted by GTG, 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are substituted by CGG, and 634th to 636th TCC of the base sequence of SEQ ID No: 4 are substituted by TAC;   (ar) 16th to 18th CTG of the base sequence of SEQ ID No: 3 are substituted by GCG, 55th to 57th GCG of the base sequence of SEQ ID No: 3 are substituted by GTG, 376th to 378th TTC of the base sequence of SEQ ID No: 3 are substituted by TAC, 379th to 381st CTG of the base sequence of SEQ ID No: 4 are substituted by TCG, 478th to 480th CGG of the base sequence of SEQ ID No: 4 are substituted by TGG, and 556th to 558th CTG of the base sequence of SEQ ID No: 4 are substituted by CGG;   (as) 16th to 18th CTG of the base sequence of SEQ ID No: 3 are substituted by ACG, 106th to 108th ACG of the base sequence of SEQ ID No: 3 are substituted by ATG, 376th to 378th TTC of the base sequence of SEQ ID No: 3 are substituted by TAC, 28th to 30th ACC of the base sequence of SEQ ID No: 4 are substituted by GAC, 352nd to 354th TTC of the base sequence of SEQ ID No: 4 are substituted by GTC, and 598th to 600th GCC of the base sequence of SEQ ID No: 4 are substituted by GAG;   (at) 55th to 57th GCG of the base sequence of SEQ ID No: 3 are substituted by GTG, 211th to 213th CGT of the base sequence of SEQ ID No: 3 are substituted by CAT, 376th to 378th TTC of the base sequence of SEQ ID No: 3 are substituted by TAC, 109th to 111th TTC of the base sequence of SEQ ID No: 4 are substituted by CTC, 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are substituted by GAT, and 598th to 600th GCC of the base sequence of SEQ ID No: 4 are substituted by GAG;   (au) 55th to 57th GCG of the base sequence of SEQ ID No: 3 are substituted by GTG, 211th to 213th CGT of the base sequence of SEQ ID No: 3 are substituted by CAT, 376th to 378th TTC of the base sequence of SEQ ID No: 3 are substituted by TAC, 109th to 111th TTC of the base sequence of SEQ ID No: 4 are substituted by GTC, 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are substituted by GAT, and 598th to 600th GCC of the base sequence of SEQ ID No: 4 are substituted by GAG;   (av) 106th to 108th ACG of the base sequence of SEQ ID No: 3 are substituted by ATG, 442nd to 444th GGC of the base sequence of SEQ ID No: 3 are substituted by GAC, 610th to 612th GTC of the base sequence of SEQ ID No: 3 are substituted by CGC, 121st to 123rd TTC of the base sequence of SEQ ID No: 4 are substituted by ATC, 151st to 153rd TTC of the base sequence of SEQ ID No: 4 are substituted by GTC, and 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are substituted by GAT;   (aw) 442nd to 444th GGC of the base sequence of SEQ ID No: 3 are substituted by GAC, 610th to 612th GTC of the base sequence of SEQ ID No: 3 are substituted by CGC, 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are substituted by GAT, and 598th to 600th GCC of the base sequence of SEQ ID No: 4 are substituted by GAG;   (ax) 106th to 108th ACG of the base sequence of SEQ ID No: 3 are substituted by GGG, and 562nd to 564th ACC of the base sequence of SEQ ID No: 3 are substituted by GGC;   (ay) 106th to 108th ACG of the base sequence of SEQ ID No: 3 are substituted by GCG, and 142nd to 144th AAC of the base sequence of SEQ ID No: 3 are substituted by CAA;   (az) 142nd to 144th AAC of the base sequence of SEQ ID No: 3 are substituted by GAA, and 436th to 438th CGG of the base sequence of SEQ ID No: 4 are substituted by GGG;   (ba) 106th to 108th ACG of the base sequence of SEQ ID No: 3 are substituted by TGG, and 526th to 528th TAC of the base sequence of SEQ ID No: 4 are substituted by TGC;   (bb) 526th to 528th TAC of the base sequence of SEQ ID No: 4 are substituted by ATG, and 649th to 651st GAC of the base sequence of SEQ ID No: 4 are substituted by GGC;   (bc) 106th to 108th ACG of the base sequence of SEQ ID No: 3 are substituted by TCG, and 97th to 99th GCG of the base sequence of SEQ ID No: 4 are substituted by GTG;   (bd) 526th to 528th TAC of the base sequence of SEQ ID No: 4 are substituted by GCC, and 649th to 651st GAC of the base sequence of SEQ ID No: 4 are substituted by GTC;   (be) 118th to 120th ACG of the base sequence of SEQ ID No: 4 are substituted by GTG, and 652nd to 654th TGC of the base sequence of SEQ ID No: 4 are substituted by ATG;   (bf) 97th to 99th GCG of the base sequence of SEQ ID No: 4 are substituted by ATG, and 526th to 528th TAC of the base sequence of SEQ ID No: 4 are substituted by ACC;   (bg) 118th to 120th ACG of the base sequence of SEQ ID No: 4 are substituted by CTG, and 649th to 651st GAC of the base sequence of SEQ ID No: 4 are substituted by CTC;   (bh) 118th to 120th ACG of the base sequence of SEQ ID No: 4 are substituted by ATT, and 181st to 183rd GCC of the base sequence of SEQ ID No: 4 are substituted by GTC;   (bi) 181st to 183rd GCC of the base sequence of SEQ ID No: 4 are substituted by ACG, and 652nd to 654th TGC of the base sequence of SEQ ID No: 4 are substituted by TCC;   (bj) 334th to 336th AAG of the base sequence of SEQ ID No: 4 are substituted by GTG, and 649th to 651st GAC of the base sequence of SEQ ID No: 4 are substituted by ATG;   (bk) 181st to 183rd GCC of the base sequence of SEQ ID No: 4 are substituted by TGG, and 649th to 651st GAC of the base sequence of SEQ ID No: 4 are substituted by CAC;   (bl) 181st to 183rd GCC of the base sequence of SEQ ID No: 4 are substituted by CTC, and 334th to 336th AAG of the base sequence of SEQ ID No: 4 are substituted by ATT;   (bm) 436th to 438th CGG of the base sequence of SEQ ID No: 4 are substituted by GGG, and 649th to 651st GAC of the base sequence of SEQ ID No: 4 are substituted by AGC;   (bn) 511th to 513th AAG of the base sequence of SEQ ID No: 4 are substituted by GCG, and 649th to 651st GAC of the base sequence of SEQ ID No: 4 are substituted by ACC;   (bo) 448th to 450th GCG of the base sequence of SEQ ID No: 4 are substituted by TCG, and 649th to 651st GAC of the base sequence of SEQ ID No: 4 are substituted by TGT;   (bp) 181st to 183rd GCC of the base sequence of SEQ ID No: 4 are substituted by GGC, and 448th to 450th GCG of the base sequence of SEQ ID No: 4 are substituted by AAT;   (bq) 181st to 183rd GCC of the base sequence of SEQ ID No: 4 are substituted by TCG, and 478th to 480th CGG of the base sequence of SEQ ID No: 4 are substituted by ATG; and   (br) 478th to 480th CGG of the base sequence of SEQ ID No: 4 are substituted by TGT, and 502nd to 504th ACG of the base sequence of SEQ ID No: 4 are substituted by GAG.   
     
     
         12 . The gene encoding a nitrile hydratase variant according to  claim 8 , comprising substitutions of the following (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII) or (XIV):
 (I) the base sequences of 106th to 108th, 274th to 276th, 442nd to 444th and 610th to 612th in SEQ ID No: 3, and the base sequences of 121st to 123rd, 151st to 153rd, and 322nd to 324th in SEQ ID No: 4;   (II) the base sequences of 16th to 18th, 55th to 57th, 280th to 282nd and 376th to 378th in SEQ ID No: 3, and the base sequences of 136th to 138th, 322nd to 324th and 634th to 636th in SEQ ID No: 4;   (III) the base sequences of 16th to 18th, 55th to 57th, and 376th to 378th in SEQ ID No: 3, and the base sequences of 10th to 12th, 379th to 381st, 478th to 480th and 556th to 558th in SEQ ID No: 4;   (IV) the base sequences of 55th to 57th, 211th to 213th and 376th to 378th in SEQ ID No: 3, and the base sequences of 109th to 111th, 235th to 237th, 322nd to 324th and 598th to 600th in SEQ ID No: 4;   (V) the base sequences of 16th to 18th, 55th to 57th, and 376th to 378th in SEQ ID No: 3, and the base sequences of 142nd to 144th, 286th to 288th, 322nd to 324th and 634th to 636th in SEQ ID No: 4;   (VI) the base sequences of 55th to 57th, 211th to 213th, and 376th to 378th in SEQ ID No: 3, and the base sequences of 109th to 111th, 319th to 321st, 322nd to 324th and 598th to 600th in SEQ ID No: 4;   (VII) the base sequences of 37th to 39th, 55th to 57th, 211th to 213th and 376th to 378th in SEQ ID No: 3, and the base sequences of 109th to 111th, 286th to 288th, 322nd to 324th and 598th to 600th in SEQ ID No: 4;   (VIII) the base sequences of 16th to 18th, 79th to 81st, 106th to 108th and 376th to 378th in SEQ ID No: 3, and the base sequences of 28th to 30th, 319th to 321st, 352nd to 354th and 598th to 600th in SEQ ID No: 4;   (IX) the base sequences of 16th to 18th, 55th to 57th and 376th to 378th in SEQ ID No: 3, and the base sequences of 142nd to 144th, 235th to 237th, 322nd to 324th, 634th to 636th and 688th to 690th in SEQ ID No: 4;   (X) the base sequences of 16th to 18th, 106th to 108th and 376th to 378th in SEQ ID No: 3, and the base sequences of 28th to 30th, 352nd to 354th, 598th to 600th, 616th to 618th and 688th to 690th in SEQ ID No: 4;   (XI) the base sequences of 106th to 108th, 422nd to 444th and 610th to 612th in SEQ ID No: 3, and the base sequences of 121st to 123rd, 151st to 153rd, 322nd to 324th, 616th to 618th and 688th to 690th in SEQ ID No: 4;   (XII) the base sequences of 16th to 18th, 55th to 57th and 376th to 378th in SEQ ID No: 3, and the base sequences of 142nd to 144th, 235th to 237th, 322nd to 324th, 634th to 636th, 688th to 690th and 691st to 693rd in SEQ ID No: 4;   (XIII) the base sequences of 16th to 18th, 55th to 57th and 376th to 378th in SEQ ID No: 3, and the base sequences of 136th to 138th, 235th to 237th, 322nd to 324th, 634th to 636th, 688th to 690th and 691st to 693rd in SEQ ID No: 4;   (XIV) the base sequences of 16th to 18th, 55th to 57th and 376th to 378th in SEQ ID No: 3, and the base sequences of 70th to 72st, 142nd to 148th, 235th to 237th, 322nd to 324th, 634th to 636th, 688th to 690th and 691st to 693rd in SEQ ID No: 4;   
     
     
         13 . The gene encoding a nitrile hydratase variant according to  claim 12 ,
 wherein in case of substitutions of (I):   106th to 108th of the base sequence of SEQ ID No: 3 are substituted by ATG;   274th to 276th of the base sequence of SEQ ID No: 3 are substituted by GAG;   442nd to 444th of the base sequence of SEQ ID No: 3 are substituted by GAC;   610th to 612th of the base sequence of SEQ ID No: 3 are substituted by CGC;   121st to 123rd of the base sequence of SEQ ID No: 4 are substituted by ATC;   151st to 153rd of the base sequence of SEQ ID No: 4 are substituted by GTC; and   322nd to 324th of the base sequence of SEQ ID No: 4 are substituted by GAT;   wherein in case of substitutions of (II):   16th to 18th of the base sequence of SEQ ID No: 3 are substituted by ACG;   55th to 57th of the base sequence of SEQ ID No: 3 are substituted by GTG;   280th to 282nd of the base sequence of SEQ ID No: 3 are substituted by ATC;   376th to 378th of the base sequence of SEQ ID No: 3 are substituted by TAC;   136th to 138th of the base sequence of SEQ ID No: 4 are substituted by AAG;   322nd to 324th of the base sequence of SEQ ID No: 4 are substituted by CGG; and   634th to 636th of the base sequence of SEQ ID No: 4 are substituted by TAC;   wherein in case of substitutions of (III):   16th to 18th of the base sequence of SEQ ID No: 3 are substituted by GCG;   55th to 57th of the base sequence of SEQ ID No: 3 are substituted by GTG;   376th to 378th of the base sequence of SEQ ID No: 3 are substituted by TAC;   10th to 12th of the base sequence of SEQ ID No: 4 are substituted by ATG;   379th to 381st of the base sequence of SEQ ID No: 4 are substituted by TCG;   478th to 480th of the base sequence of SEQ ID No: 4 are substituted by TGG; and   556th to 558th of the base sequence of SEQ ID No: 4 are substituted by CGG;   wherein in case of substitutions of (IV):   55th to 57th of the base sequence of SEQ ID No: 3 are substituted by GTG;   211th to 213th of the base sequence of SEQ ID No: 3 are substituted by CAT;   376th to 378th of the base sequence of SEQ ID No: 3 are substituted by TAC;   109th to 111th of the base sequence of SEQ ID No: 4 are substituted by GTC;   235th to 237th of the base sequence of SEQ ID No: 4 are substituted by AAC;   322nd to 324th of the base sequence of SEQ ID No: 4 are substituted by GAT; and   598th to 600th of the base sequence of SEQ ID No: 4 are substituted by GAG;   wherein in case of substitutions of (V):   16th to 18th of the base sequence of SEQ ID No: 3 are substituted by ACG;   55th to 57th of the base sequence of SEQ ID No: 3 are substituted by GTG;   376th to 378th of the base sequence of SEQ ID No: 3 are substituted by TAC;   142nd to 144th of the base sequence of SEQ ID No: 4 are substituted by GTG;   286th to 288th of the base sequence of SEQ ID No: 4 are substituted by CGT;   322nd to 324th of the base sequence of SEQ ID No: 4 are substituted by CGG; and   634th to 636th of the base sequence of SEQ ID No: 4 are substituted by TAC;   wherein in case of substitutions of (VI):   55th to 57th of the base sequence of SEQ ID No: 3 are substituted by GTG;   211th to 213th of the base sequence of SEQ ID No: 3 are substituted by CAT;   376th to 378th of the base sequence of SEQ ID No: 3 are substituted by TAC;   109th to 111th of the base sequence of SEQ ID No: 4 are substituted by GTC;   319th to 321st of the base sequence of SEQ ID No: 4 are substituted by ATG;   322nd to 324th of the base sequence of SEQ ID No: 4 are substituted by GAT; and   598th to 600th of the base sequence of SEQ ID No: 4 are substituted by GAG;   wherein in case of substitutions of (VII):   37th to 39th of the base sequence of SEQ ID No: 3 are substituted by CTC;   55th to 57th of the base sequence of SEQ ID No: 3 are substituted by GTG;   211th to 213th of the base sequence of SEQ ID No: 3 are substituted by CAT;   376th to 378th of the base sequence of SEQ ID No: 3 are substituted by TAC;   109th to 111th of the base sequence of SEQ ID No: 4 are substituted by CTC;   286th to 288th of the base sequence of SEQ ID No: 4 are substituted by CGT;   322th to 324th of the base sequence of SEQ ID No: 4 are substituted by GAT; and   598th to 600th of the base sequence of SEQ ID No: 4 are substituted by GAG;   wherein in case of substitutions of (VIII):   16th to 18th of the base sequence of SEQ ID No: 3 are substituted by ACG;   79th to 81st of the base sequence of SEQ ID No: 3 are substituted by ATC;   106th to 108th of the base sequence of SEQ ID No: 3 are substituted by ATG;   376th to 378th of the base sequence of SEQ ID No: 3 are substituted by TAC;   28th to 30th of the base sequence of SEQ ID No: 4 are substituted by GAC;   319th to 321st of the base sequence of SEQ ID No: 4 are substituted by ATG;   352nd to 354th of the base sequence of SEQ ID No: 4 are substituted by GTC; and   598th to 600th of the base sequence of SEQ ID No: 4 are substituted by GAG;   wherein in case of substitutions of (IX):   16th to 18th of the base sequence of SEQ ID No: 3 are substituted by ACG;   55th to 57th of the base sequence of SEQ ID No: 3 are substituted by GTG;   376th to 378th of the base sequence of SEQ ID No: 3 are substituted by TAC;   142nd to 144th of the base sequence of SEQ ID No: 4 are substituted by GTG;   235th to 237th of the base sequence of SEQ ID No: 4 are substituted by AAC;   322nd to 324th of the base sequence of SEQ ID No: 4 are substituted by CGG;   634th to 636th of the base sequence of SEQ ID No: 4 are substituted by TAC; and   688th to 690th of the base sequence of SEQ ID No: 4 are substituted by GAG;   wherein in case of substitutions of (X):   16th to 18th of the base sequence of SEQ ID No: 3 are substituted by ACG;   106th to 108th of the base sequence of SEQ ID No: 3 are substituted by ATG;   376th to 378th of the base sequence of SEQ ID No: 3 are substituted by TAC;   28th to 30th of the base sequence of SEQ ID No: 4 are substituted by GAC;   352nd to 354th of the base sequence of SEQ ID No: 4 are substituted by GTC;   598th to 600th of the base sequence of SEQ ID No: 4 are substituted by GAG;   616th to 618th of the base sequence of SEQ ID No: 4 are substituted by CTG; and   688th to 690th of the base sequence of SEQ ID No: 4 are substituted by GAG;   wherein in case of substitutions of (XI):   106th to 108th of the base sequence of SEQ ID No: 3 are substituted by ATG;   442nd to 444th of the base sequence of SEQ ID No: 3 are substituted by GAC;   610th to 612th of the base sequence of SEQ ID No: 3 are substituted by CGC;   121st to 123rd of the base sequence of SEQ ID No: 4 are substituted by ATC;   151st to 153rd of the base sequence of SEQ ID No: 4 are substituted by GTC;   322nd to 324th of the base sequence of SEQ ID No: 4 are substituted by GAT;   616th to 618th of the base sequence of SEQ ID No: 4 are substituted by CTG; and   688th to 690th of the base sequence of SEQ ID No: 4 are substituted by GAG;   wherein in case of substitutions of (XII):   16th to 18th of the base sequence of SEQ ID No: 3 are substituted by ACG;   55th to 57th of the base sequence of SEQ ID No: 3 are substituted by GTG;   376th to 378th of the base sequence of SEQ ID No: 3 are substituted by TAC;   142nd to 144th of the base sequence of SEQ ID No: 4 are substituted by GTG;   235th to 237th of the base sequence of SEQ ID No: 4 are substituted by AAC;   322nd to 324th of the base sequence of SEQ ID No: 4 are substituted by CGG;   634th to 636th of the base sequence of SEQ ID No: 4 are substituted by TAC;   688th to 690th of the base sequence of SEQ ID No: 4 are substituted by GAG; and   691st to 693rd of the base sequence of SEQ ID No: 4 are substituted by GTC;   wherein in case of substitutions of (XIII):   16th to 18th of the base sequence of SEQ ID No: 3 are substituted by ACG;   55th to 57th of the base sequence of SEQ ID No: 3 are substituted by GTG;   376th to 378th of the base sequence of SEQ ID No: 3 are substituted by TAC;   136th to 138th of the base sequence of SEQ ID No: 4 are substituted by AAG;   235th to 237th of the base sequence of SEQ ID No: 4 are substituted by AAC;   322nd to 324th of the base sequence of SEQ ID No: 4 are substituted by CGG;   634th to 636th of the base sequence of SEQ ID No: 4 are substituted by TAC;   688th to 690th of the base sequence of SEQ ID No: 4 are substituted by GAG; and   691st to 693rd of the base sequence of SEQ ID No: 4 are substituted by GTC;   wherein in case of substitutions of (XIV):   16th to 18th of the base sequence of SEQ ID No: 3 are substituted by ACG;   55th to 57th of the base sequence of SEQ ID No: 3 are substituted by GTG;   376th to 378th of the base sequence of SEQ ID No: 3 are substituted by TAC;   70th to 72st of the base sequence of SEQ ID No: 4 are substituted by ATC;   142nd to 148th of the base sequence of SEQ ID No: 4 are substituted by GTG;   235th to 237th of the base sequence of SEQ ID No: 4 are substituted by AAC;   322nd to 324th of the base sequence of SEQ ID No: 4 are substituted by CGG;   634th to 636th of the base sequence of SEQ ID No: 4 are substituted by TAC;   688th to 690th of the base sequence of SEQ ID No: 4 are substituted by GAG; and   691st to 693rd of the base sequence of SEQ ID No: 4 are substituted by GTC.   
     
     
         14 . A linked DNA comprising further DNA containing a promoter sequence necessary for the expression of the gene in the upstream region of the 5′-terminal of the gene encoding a nitrile hydratase variant according to  claim 8 , and a ribosome binding sequence contained in SEQ ID No: 7 in the downstream region of the 3′-terminal of the promoter. 
     
     
         15 . A plasmid comprising the DNA according to  claim 14 . 
     
     
         16 . A transformant obtained by transformation of a host cell using the plasmid according to  claim 15 . 
     
     
         17 . A method for producing a nitrile hydratase variant, comprising cultivating the transformant according to  claim 16  in a culture medium and producing a nitrile hydratase variant based on the nitrile hydratase gene carried by the plasmid in the transformant.