Nitrilases, nucleic acids encoding them and methods for making and using them

Patent metadata

Field	Value
Publication number	US-2016355801-A1
Application number	US-201615064269-A
Country	US
Kind code	A1
Filing date	Mar 8, 2016
Priority date	Dec 29, 1999
Publication date	Dec 8, 2016
Grant date	—

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.

First claim

What is claimed is:

1 - 13 . (canceled)

14 . An isolated, synthetic or recombinant nucleic acid, encoding a polypeptide having nitrilase activity, comprising:
(a) a nucleic acid having a sequence at least 85% identical to 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 or 385; or (b) nucleic acid encoding a polypeptide comprising consecutive amino acids having a sequence at least 85% identical to 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 or 386; or (c) a nucleic acid completely complementary to (a) or (b).

15 . A nucleic acid vector capable of replication in a host cell, wherein the vector comprises the nucleic acid of claim 14 .

16 . A host cell comprising the nucleic acid of claim 14 .

17 . A host organism comprising the host cell of claim 16 .

18 . The isolated, synthetic or recombinant nucleic acid of claim 14 , wherein the nucleic acid is affixed to a solid support, wherein optionally the solid support is selected from the group of a gel, a resin, a polymer, a ceramic, a glass, a microelectrode and any combination thereof.

19 . A method for making a polypeptide having nitrilase activity, the method comprising (a) introducing the nucleic acid of claim 14 encoding the polypeptide into a host cell under conditions that permit production of the polypeptide by the host cell, and (b) recovering the recombinant polypeptide so produced.

20 . A kit comprising the nucleic acid of claim 14 , or a fragment thereof encoding a polypeptide having nitrilase activity, and a buffer.

21 . The isolated, synthetic or recombinant nucleic acid of claim 14 , further consisting of one or more mutations, wherein: nucleotides 163-165 are substituted with AAA, AAG, GGT, GGC, GGA, GGG, CAA, or CAG; nucleotides 178-180 are substituted with GAA or GAG; nucleotides 331-333 are substituted with TCT, TCC, TCA, TCG, AGT, or AGC; nucleotides 568-570 are substituted with CAT, CAC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, TCA, TAT, TAC, ATG or ACG; nucleotides 571-573 are substituted with TTA, TTG, CTT, CTC, CTA, CTG, GTT, GTC, GTA, GTG, ATG, ACT, ACC, ACA, GAT, GAC, GGT, GGC, GGA, GGG, GAA, GAG, TAT, TAC, or ACG; nucleotides 595-597 are substituted with GAA, GAG, TTA, TTG, CTT, CTC, CTA, or CTG; nucleotides 664-666 of SEQ ID NO: 209 are substituted with TTA, TTG, CTT, CTC, CTA, or CTG; or any combination thereof.

22 . An isolated, synthetic or recombinant polypeptide comprising consecutive amino acids having a sequence at least 85% identical to 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 or 386, wherein the polypeptide has nitrilase activity.

23 . A codon-optimized polypeptide of claim 22 , or a fragment thereof, having nitrilase activity, wherein the codon usage is optimized for a particular organism or cell.

24 . The polypeptide of claim 22 , or a fragment thereof, having nitrilase activity, or a peptidomimetic thereof having nitrilase activity, wherein the polypeptide, fragment, or peptidomimetic is affixed to a solid support, wherein the solid support is selected from the group consisting of a gel, a resin, a polymer, a ceramic, a glass, a microelectrode and any combination thereof.

25 . The polypeptide of claim 22 , wherein the nitrilase activity comprises hydrolyzing a nitrile to a carboxylic acid.

26 . The polypeptide of claim 22 , wherein the nitrilase activity comprises hydrolyzing a cyanohydrin moiety or an aminonitrile moiety of a molecule.

27 . A method for making a chiral alpha-hydroxy acid molecule or a chiral amino acid molecule, the method comprising admixing a molecule having a cyanohydrin moiety or an aminonitrile moiety with at least one polypeptide having an amino acid sequence at least 85% identical to any one of SEQ ID NOS: 1-386 or a fragment thereof, or a peptidomimetic thereof, having enantio-selective nitrilase activity.

28 . A method for making a composition or an intermediate thereof, the method comprising admixing a precursor of the composition or intermediate, wherein the precursor comprises a cyanohydrin moiety or an aminonitrile moiety, with at least one polypeptide of claim 22 , or a fragment or peptidomimetic thereof having nitrilase activity, hydrolyzing the cyanohydrin or the aminonitrile moiety in the precursor thereby making the composition or the intermediate thereof.

29 . An isolated, synthetic, or recombinant variant polypeptide having a nitrilase activity wherein the variant polypeptide is an amino acid sequence 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,

Assignees

• Basf Enzymes Llc

Inventors

• Wong Kelvin
• Short Jay M
• Burk Mark J
• Desantis Grace
• Farwell Robert
• Chatman Kelly

Classifications

• C12N9/78 Primary

  acting on carbon to nitrogen bonds other than peptide bonds (3.5) · CPC title

• C12P7/42

  Hydroxy-carboxylic acids · CPC title

• C12P13/002

  Nitriles (-CN) · CPC title

• C12Y305/05001

  Nitrilase (3.5.5.1) · CPC title

• C12P41/006

  by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures · CPC title