Nitrilases
OwnerGrace Desantis, Jennifer Ann Chaplin +7inventors
InventorsGrace Desantis, Jennifer Ann Chaplin, Ellen Chi, Aileen Milan, Jay M. Short, David Weiner, Mark J. Burk, Eric J. Mathur, Darcy Madden (Heir)
Life of this patent
Estimating the term…
Abstract
The invention relates to nitrilases and to nucleic acids encoding the nitrilases. In addition methods of designing new nitrilases and method of use thereof are also provided. The nitrilases have increased activity and stability at increased pH and temperature.
Claims54
An isolated nucleic acid comprising nucleotides having a sequence at least 50% identical to SEQ ID NO:
- 209, variants of SEQ ID NO:209 having one or more mutations:
- at positions 163-165 AAA, AAG, GGT, GGC, GGA, GGG, or CAA, CAG;
- at positions 178-180 GAA or GAG;
- at positions 331-333 TCT TCC, TCA, TCG, AGT, or AGC;
- at positions 568-570 CAT, CAC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, or ACG;
- at positions 571-573 TTA, TTG, CTT, CTC, CTA, CTG, GTT, GTC, GTA, GTG, ATG, ACT, ACC, ACA, or ACG;
- at positions 595-597 GAA, GAG, TTA, TTG, CTT, CTC, CTA, or CTG;
- at positions 664-666 TTA, TTG, CTT, CTC, CTA, or CTG;
- or any combination thereof, fragments thereof, wherein the nucleic acid or fragment encodes a polypeptide having nitrilase activity, or their complements.
An isolated nucleic acid comprising nucleotides having a sequence identical to the SEQ ID NO:209, fragments having nitrilase activity, or their complements.
An isolated nucleic acid comprising nucleotides having a sequence identical to a variant of SEQ ID NO:209 having one or more mutations:
- at positions 163-165 AAA, AAG, GGT, GGC, GGA, GGG, or CAA, CAG;
- at positions 178-180 GAA or GAG;
- at positions 331-333 TCT TCC, TCA, TCG, AGT, or AGC;
- at positions 568-570 CAT, CAC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, or ACG;
- at positions 571-573 TTA, TTG, CTT, CTC, CTA, CTG, GTT, GTC, GTA, GTG, ATG, ACT, ACC, ACA, or ACG;
- at positions 595-597 GAA, GAG, TTA, TTG, CTT, CTC, CTA, or CTG;
- at positions 664-666 TTA, TTG, CTT, CTC, CTA, or CTG;
- or any combination thereof, fragments having nitrilase activity, or their complements.
An isolated nucleic acid that hybridizes to a nucleic acid of SEQ ID NO:209 or variants of SEQ ID NO:209 having one or more mutations:
- at positions 163-165 AAA, AAG, GGT, GGC, GGA, GGG, or CAA, CAG;
- at positions 178-180 GAA or GAG;
- at positions 331-333 TCT TCC, TCA, TCG, AGT, or AGC;
- at positions 568-570 CAT, CAC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, or ACG;
- at positions 571-573 TTA, TTG, CTT, CTC, CTA, CTG, GTT, GTC, GTA, GTG, ATG, ACT, ACC, ACA, or ACG;
- at positions 595-597 GAA, GAG, TTA, TTG, CTT, CTC, CTA, or CTG;
- at positions 664-666 TTA, TTG, CTT, CTC, CTA, or CTG;
- or any combination thereof, fragments having nitrilase activity, or their complements.
A nucleic acid probe comprising from about 15 nucleotides to about 50 nucleotides, wherein at least 15 consecutive nucleotides are at least 50% complementary to a nucleic acid target region within a nucleic acid sequence of SEQ ID NO:209 or variants of SEQ ID NO:209 having one or more mutations:
- at positions 163-165 AAA, AAG, GGT, GGC, GGA, GGG, or CAA, CAG;
- at positions 178-180 GAA or GAG;
- at positions 331-333 TCT TCC, TCA, TCG, AGT, or AGC;
- at positions 568-570 CAT, CAC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, or ACG;
- at positions 571-573 TTA, TTG, CTT, CTC, CTA, CTG, GTT, GTC, GTA, GTG, ATG, ACT, ACC, ACA, or ACG;
- at positions 595-597 GAA, GAG, TTA, TTG, CTT, CTC, CTA, or CTG;
- at positions 664-666 TTA, TTG, CTT, CTC, CTA, or CTG;
- or any combination thereof, or their complements.
A nucleic acid probe comprising at least 15 consecutive nucleotides of a nucleic acid target region within a nucleic acid sequence of SEQ ID NO:209 or variants of SEQ ID NO:209 having one or more mutations:
- at positions 163-165 AAA, AAG, GGT, GGC, GGA, GGG, or CAA, CAG;
- at positions 178-180 GAA or GAG;
- at positions 331-333 TCT TCC, TCA, TCG, AGT, or AGC;
- at positions 568-570 CAT, CAC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, or ACG;
- at positions 571-573 TTA, TTG, CTT, CTC, CTA, CTG, GTT, GTC, GTA, GTG, ATG, ACT, ACC, ACA, or ACG;
- at positions 595-597 GAA, GAG, TTA, TTG, CTT, CTC, CTA, or CTG;
- at positions 664-666 TTA, TTG, CTT, CTC, CTA, or CTG;
- or any combination thereof, or their complements.
A nucleic acid vector capable of replication in a host cell, wherein the vector comprises the nucleic acid of any one of claims 1 to 6, 12, and 13.
A host cell comprising the nucleic acid of any one of claims 1 to 6, 12 and 13.
An isolated nucleic acid encoding a polypeptide comprising amino acids having a sequence at least 50% identical to SEQ ID NO:210 or variants of SEQ ID NO:210 having one or more mutations:
- at residue 55 lysine, glycine, or glutamine;
- at residue 60 glutamic acid;
- at residue 111 serine, at residue 190 methionine, serine, or threonine;
- at residue 191, leucine, valine, methionine, or threonine;
- at residue 199 glutamic acid or leucine;
- at residue 222 leucine;
- or any combination thereof, fragments encoding polypeptides wherein the polypeptides have nitrilase activity, or its complement.
An isolated nucleic acid encoding a polypeptide comprising amino acids having a sequence of SEQ ID NO:210 or variants of SEQ ID NO:210 having one or more mutations:
- at residue 55 lysine, glycine, or glutamine;
- at residue 60 glutamic acid;
- at residue 111 serine, at residue 190 methionine, serine, or threonine;
- at residue 191, leucine, valine, methionine, or threonine;
- at residue 199 glutamic acid or leucine;
- at residue 222 leucine;
- or any combination thereof, fragments encoding a polypeptides having nitrilase activity, or its complement.
An isolated polypeptide comprising amino acids having a sequence at least 50% identical to SEQ ID NO:210, or variants of SEQ ID NO:210 having one or more mutations:
- at residue 55 lysine, glycine, or glutamine;
- at residue 60 glutamic acid;
- at residue 111 serine, at residue 190 methionine, serine, or threonine;
- at residue 191, leucine, valine, methionine, or threonine;
- at residue 199 glutamic acid or leucine;
- at residue 222 leucine;
- or any combination thereof, or fragments thereof, wherein the polypeptide has nitrilase activity.
An isolated polypeptide comprising amino acid having SEQ ID NO:210 or variants of SEQ ID NO:210 having one or more mutations:
- at residue 55 lysine, glycine, or glutamine;
- at residue 60 glutamic acid;
- at residue 111 serine, at residue 190 methionine, serine, or threonine;
- at residue 191, leucine, valine, methionine, or threonine;
- at residue 199 glutamic acid or leucine;
- at residue 222 leucine;
- or any combination thereof, or fragments thereof, wherein the polypeptide has nitrilase activity.
A method for making an (R)-ethyl 4-cyano-3-hydroxybutyric acid, the method comprising contacting a hydroxyglutaryl nitrile with at least one polypeptide encoded by a nucleic acid having a sequence of SEQ ID NO:209 or a variant of SEQ ID NO:209 having one or more mutations:
- at positions 163-165 AAA, AAG, GGT, GGC, GGA, GGG, or CAA, CAG;
- at positions 178-180 GAA or GAG;
- at positions 331-333 TCT TCC, TCA, TCG, AGT, or AGC;
- at positions 568-570 CAT, CAC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, or ACG;
- at positions 571-573 TTA, TTG, CTT, CTC, CTA, CTG, GTT, GTC, GTA, GTG, ATG, ACT, ACC, ACA, or ACG;
- at positions 595-597 GAA, GAG, TTA, TTG, CTT, CTC, CTA, or CTG;
- at positions 664-666 TTA, TTG, CTT, CTC, CTA, or CTG;
- or any combination thereof, or a fragment thereof encoding a polypeptide having nitrilase activity, that selectively produces an (R)-enantiomer, so as to make (R)-ethyl 4-cyano-3-hydroxybutyric acid.
A method for making an (S)-ethyl 4-cyano-3-hydroxybutyric acid, the method comprising contacting a hydroxyglutaryl nitrile with at least one polypeptide having an amino acid sequence of any one of SEQ ID NO:210 or a variant of SEQ ID NO:210 having one or more mutations:
- at residue 55 lysine, glycine, or glutamine;
- at residue 60 glutamic acid;
- at residue 111 serine, at residue 190 methionine, serine, or threonine;
- at residue 191, leucine, valine, methionine, or threonine;
- at residue 199 glutamic acid or leucine;
- at residue 222 leucine;
- or any combination thereof, or a fragment or peptidomimetic thereof having nitrilase activity that selectively produces an (S)-enantiomer, so as to make (S)-ethyl 4-cyano-3-hydroxybutyric acid.
A method for making an (R)-mandelic acid, the method comprising admixing a mandelonitrile with at least one polypeptide having an amino acid sequence of any one of SEQ ID NO:210 or a variant of SEQ ID NO:210 having one or more mutations:
- at residue 55 lysine, glycine, or glutamine;
- at residue 60 glutamic acid;
- at residue 111 serine, at residue 190 methionine, serine, or threonine;
- at residue 191, leucine, valine, methionine, or threonine;
- at residue 199 glutamic acid or leucine;
- at residue 222 leucine;
- or any combination thereof, or any fragment or peptidomimetic thereof having nitrilase activity.
A method for making an (S)-mandelic acid, the method comprising admixing a mandelonitrile with at least one polypeptide having an amino acid sequence of SEQ ID NO:210 or a variant of SEQ ID NO:210 having one or more mutations:
- at residue 55 lysine, glycine, or glutamine;
- at residue 60 glutamic acid;
- at residue 111 serine, at residue 190 methionine, serine, or threonine;
- at residue 191, leucine, valine, methionine, or threonine;
- at residue 199 glutamic acid or leucine;
- at residue 222 leucine;
- or any combination thereof, or any fragment or peptidomimetic thereof having nitrilase activity.
A method for making an (S)-phenyl lactic acid derivative or an (R)-phenyl lactic acid derivative, the method comprising admixing a phenyllactocyanonitrile with at least one polypeptide selected from SEQ ID NO:210 or a variant of SEQ ID NO:210 having one or more mutations:
- at residue 55 lysine, glycine, or glutamine;
- at residue 60 glutamic acid;
- at residue 111 serine, at residue 190 methionine, serine, or threonine;
- at residue 191, leucine, valine, methionine, or threonine;
- at residue 199 glutamic acid or leucine;
- at residue 222 leucine;
- or any combination thereof, or any fragment or peptidomimetic thereof having nitrilase activity, or any active fragment or peptidomimetic thereof that selectively produces an (S)-enantiomer or an (R)-enantiomer, thereby producing an (S)-phenyl lactic acid derivative or an (R)-phenyl lactic acid derivative.
A computer readable medium having stored thereon at least one nucleotide sequence selected from the group consisting of:
- SEQ ID NO:
- 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383 385, or variants thereof, and/or at least one amino acid sequence selected from the group consisting of:
- SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384 and 386, and variants thereof.
A computer system comprising a processor and a data storage device, wherein the data storage device has stored thereon at least one nucleotide sequence selected from the group consisting of SEQ ID NO:
- 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, and variants thereof and/or at least one amino acid sequence selected from the group consisting of:
- SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, and variants thereof.
A method for identifying a feature in a sequence which comprises:
- (a) inputting the sequence into a computer;
- (b) running a sequence feature identification program on the computer so as to identify a feature within the sequence;
- and
- (c) identifying the feature in the sequence, wherein the sequence comprises SEQ ID NOS:
- 1-386, variants, or any combination thereof.
An assay for screening enantioselective transformation comprising:
- (a) labeling one of two prochiral or enantiotopic moieties in a molecule;
- (b) modifying at least one of the two moieties by selective catalyst to produce products;
- and
- (c) determining the resultant products by mass spectroscopy.
Dependencies are inferred from the claim text.