US2009038023A1PendingUtilityA1
Lyase Enzymes, Nucleic Acids Encoding Them and Methods For Making and Using Them
Est. expiryMar 10, 2025(expired)· nominal 20-yr term from priority
C12Y 403/01005C12P 13/24C12P 13/222C12P 13/225C12P 7/42C12N 9/88A61P 3/00C12Y 403/01023D06M 16/003
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Claims
Abstract
This invention provides polypeptides having lyase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides having ammonia lyase activity, e.g., phenylalanine ammonia lyase, tyrosine ammonia lyase and/or histidine ammonia lyase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural and industrial contexts. X═NO 2 , Cl, Br, F, NH 2 , OH, H, alkyl at one or several o, m, and p positions R═H or alkyl
Claims
exact text as granted — not AI-modified1 . An isolated or recombinant nucleic acid comprising
(a) a nucleic acid sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249 or SEQ ID NO:251, over a region of at least about 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or more residues, wherein the nucleic acid encodes at least one polypeptide having a lyase activity, or encodes a peptide or polypeptide that can be used to generate an antibody that specifically binds to a polypeptide having a sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250 or SEQ ID NO:252, and optionally the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection; or (b) a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:01, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249 or SEQ ID NO:251, wherein the nucleic acid encodes a polypeptide having a lyase activity, or encodes a peptide or polypeptide that can be used to generate an antibody that specifically binds to a polypeptide having a sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:10, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250 or SEQ ID NO:252, and the stringent conditions include a wash step comprising a wash in 0.2×SSC at a temperature of about 65° C. for about 15 minutes, and optionally the nucleic acid is at least about 20, 25, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more residues in length or the full length of the gene or transcript; (c) a nucleic acid sequence encoding a polypeptide having a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250 or SEQ ID NO:252; or (d) a nucleic acid sequence complementary to (a), (b) or (c).
2 . The isolated or recombinant nucleic acid of claim 1 , wherein the nucleic acid sequence comprises a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:11, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249 or SEQ ID NO:251.
3 . The isolated or recombinant nucleic acid of claim 1 , wherein the sequence comparison algorithm is a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall-p blastp-d “nr pataa”-F F, and all other options are set to default.
4 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity comprises an ammonia lyase activity.
5 . The isolated or recombinant nucleic acid of claim 4 , wherein the ammonia lyase activity comprises a phenylalanine ammonia lyase activity.
6 . The isolated or recombinant nucleic acid of claim 4 , wherein the ammonia lyase activity comprises catalyzing the deamination of phenylalanine.
7 . The isolated or recombinant nucleic acid of claim 4 , wherein the ammonia lyase activity comprises tyrosine ammonia lyase activity.
8 . The isolated or recombinant nucleic acid of claim 4 , wherein the lyase activity comprises catalyzing the deamination of tyrosine.
9 . The isolated or recombinant nucleic acid of claim 4 , wherein the ammonia lyase activity comprises histidine ammonia lyase activity.
10 . The isolated or recombinant nucleic acid of claim 9 , wherein the histidine ammonia lyase activity comprises generation of urocanoic acid or urocanoic acid derivatives.
11 . The isolated or recombinant nucleic acid of claim 4 , wherein the lyase activity comprises catalyzing the deamination of histidine.
12 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity comprises catalysis of the deamination of phenylalanine, tyrosine, histidine or derivatives thereof to generate cinnamic acid, para-hydroxycinnamic acid, para-hydroxyl styrene or derivatives thereof.
13 . The isolated or recombinant nucleic acid of claim 4 , wherein the ammonia lyase activity comprises phenylalanine ammonia lyase, histidine ammonia lyase or tyrosine ammonia lyase activity or any combination thereof.
14 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity comprises catalysis of the deamination of phenylalanine, tyrosine, histidine or derivatives thereof to generate L- or D-β-amino acids, or derivatives thereof.
15 . The isolated or recombinant nucleic acid of claim 14 , wherein the lyase activity comprises catalysis of the deamination of histidine or derivatives thereof to generate L- and D-β-histidine, or derivatives thereof.
16 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity comprises catalyzing the deamination of phenylalanine, tyrosine or histidine to generate a 3,5-dihydro-5-methylidine-4H-imidazol-4-one (MIO).
17 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity comprises carboxy-lyase activity.
18 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity comprises argininosuccinate lyase activity.
19 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity comprises hydroxynitrile lyase activity.
20 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity comprises C17,20-lyase activity.
21 . The isolated or recombinant nucleic acid of claim 20 , wherein the lyase substrate is progesterone or 17-hydroxyprogesterone.
22 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity comprises dimethylsulphoniopropionate (DMSP) lyase activity.
23 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity comprises 5′-deoxyribose phosphate lyase activity.
24 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity comprises isocitrate lyase activity, catalyzing the formation of succinate and glyoxylate from isocitrate.
25 . The isolated or recombinant nucleic acid of claim 24 wherein the lyase activity comprises oxalomalate lyase activity, catalyzing the formation of oxaloacetate and glyoxylate.
26 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity comprises hydroxymethylglutaryl-CoA lyase activity.
27 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity is thermostable.
28 . The isolated or recombinant nucleic acid of claim 27 , wherein the polypeptide retains a lyase activity under conditions comprising a temperature range of between about 37° C. to about 95° C., or between about 55° C. to about 85° C., or between about 70° C. to about 75° C., or between about 70° C. to about 95° C., or between about 90° C. to about 95° C., or retains a lyase activity in a temperature in the range between about 1° C. to about 5° C., between about 5° C. to about 15° C., between about 15° C. to about 25° C., between about 25° C. to about 37° C., or between about 37° C. to about 95° C., 96° C., 97° C., 98° C. or 99° C.
29 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity is thermotolerant.
30 . The isolated or recombinant nucleic acid of claim 29 , wherein the polypeptide retains a lyase activity after exposure to a temperature in the range from greater than 37° C. to about 95° C., from greater than 55° C. to about 85° C., or between about 70° C. to about 75° C., or from greater than 90° C. to about 95° C., or after exposure to a temperature in the range between about 1° C. to about 5° C., between about 5° C. to about 15° C., between about 15° C. to about 25° C., between about 25° C. to about 37° C., or between about 37° C. to about 95° C., 96° C., 97° C., 98° C. or 99° C.
31 . A nucleic acid probe for identifying a nucleic acid encoding a polypeptide with a lyase activity, wherein the probe comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600 or more consecutive bases of a sequence as set forth in claim 1 , wherein the probe identifies the nucleic acid by binding or hybridization,
wherein optionally the probe comprises an oligonucleotide comprising at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, about 60 to 100, or about 50 to 150 consecutive bases, wherein optionally the probe comprises consecutive bases of a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249 or SEQ ID NO:251.
32 . An amplification primer pair for amplifying a nucleic acid encoding a polypeptide having a lyase activity, wherein the amplification primer pair is capable of amplifying a nucleic acid comprising a sequence of claim 1 , or a subsequence thereof, and the amplification primer pair comprises
(i) a first member having a sequence as set forth by about the first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more residues of a sequence of claim 1 ; and (ii) and a second member having a sequence as set forth by about the first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more residues of the complementary strand of the first member, wherein optionally a member of the amplification primer pair comprises an oligonucleotide comprising at least about 10 to 50 consecutive bases of the sequence, or, about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive bases of the sequence, and optionally the first member has a sequence as set forth by about the first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more residues of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249 or SEQ ID NO:251.
33 . A lyase-encoding nucleic acid generated by amplification of a polynucleotide using an amplification primer pair as set forth in claim 32 , wherein optionally the amplification is by polymerase chain reaction (PCR).
34 . The lyase-encoding nucleic acid of claim 33 , wherein the nucleic acid generated by amplification of a gene library, and optionally the gene library is an environmental library.
35 . An isolated or recombinant lyase encoded by the lyase-encoding nucleic acid set forth in claim 33 .
36 . A method of amplifying a nucleic acid encoding a polypeptide having a lyase activity comprising amplification of a template nucleic acid with an amplification primer pair as set forth in claim 32 .
37 . An expression cassette comprising a nucleic acid comprising a sequence as set forth in claim 1 .
38 . A vector comprising a nucleic acid comprising a sequence as set forth in claim 1 , wherein optionally the vehicle comprises an expression vector.
39 . A cloning vehicle comprising a nucleic acid comprising a sequence as set forth in claim 1 ,
wherein optionally the cloning vehicle comprises a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificial chromosome, and optionally the viral vector comprises an adenovirus vector, a retroviral vector or an adeno-associated viral vector, and optionally the cloning vehicle comprises a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).
40 . A transformed cell comprising a nucleic acid comprising a sequence as set forth in claim 1 , or an expression cassette as set forth in claim 37 , the vector of claim 38 , or a cloning vehicle as set forth in claim 39 ,
wherein optionally the cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.
41 . A transgenic non-human animal comprising a sequence as set forth in claim 1 , wherein optionally the transgenic non-human animal is a mouse or a rat.
42 . A transgenic plant comprising a sequence as set forth in claim 1 ,
wherein optionally the plant is a corn plant, a sorghum plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed plant, a soybean plant, a rice plant, a barley plant, a grass, or a tobacco plant.
43 . A transgenic seed comprising a sequence as set forth in claim 1 ,
wherein optionally the seed is a corn seed, a wheat kernel, an oilseed, a rapeseed, a soybean seed, a palm kernel, a sunflower seed, a sesame seed, a rice, a barley, a peanut or a tobacco plant seed.
44 . An antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a sequence as set forth in claim 1 , or a subsequence thereof,
wherein optionally the antisense oligonucleotide has a length of between about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 bases.
45 . A method of inhibiting the translation of a lyase message in a cell comprising administering to the cell or expressing in the cell an antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a sequence as set forth in claim 1 .
46 . A double-stranded interference RNA (RNAi) molecule comprising a subsequence of a sequence as set forth in claim 1 ,
wherein optionally the RNAi comprises an siRNA or an miRNA, and optionally the RNAi molecule is about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more duplex nucleotides in length.
47 . A method of inhibiting the expression of a lyase in a cell comprising administering to the cell or expressing in the cell a double-stranded interference RNA (RNAi) molecule as set forth in claim 46 .
48 . An isolated or recombinant polypeptide
(i) having lyase activity and an amino acid sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or 100% sequence identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250 or SEQ ID NO:252, over a region of at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 100, 150, 200, 250, 300 or more residues, wherein optionally the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection, and optionally the sequence comparison algorithm is a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall-p blastp-d “nr pataa”-F F, and all other options are set to default; (ii) an amino acid sequence encoded by a nucleic acid as set forth in claim 1 , wherein the polypeptide has a lyase activity or has immunogenic activity in that it is capable of generating an antibody that specifically binds to a polypeptide having a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:10, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250 or SEQ ID NO:252; or (iii) an amino acid sequence as set forth in (i) or (ii), or a polypeptide encoded by a nucleic acid as set forth in claim 1 , and comprising at least one amino acid residue conservative substitution and retaining its lyase activity or immunogenic activity, wherein optionally conservative substitution comprises replacement of an aliphatic amino acid with another aliphatic amino acid; replacement of a serine with a threonine or vice versa; replacement of an acidic residue with another acidic residue; replacement of a residue bearing an amide group with another residue bearing an amide group; exchange of a basic residue with another basic residue; or, replacement of an aromatic residue with another aromatic residue, or a combination thereof, and optionally the aliphatic residue comprises Alanine, Valine, Leucine, Isoleucine or a synthetic equivalent thereof; the acidic residue comprises Aspartic acid, Glutamic acid or a synthetic equivalent thereof, the residue comprising an amide group comprises Aspartic acid, Glutamic acid or a synthetic equivalent thereof; the basic residue comprises Lysine, Arginine or a synthetic equivalent thereof; or, the aromatic residue comprises Phenylalanine, Tyrosine or a synthetic equivalent thereof.
49 . The isolated or recombinant polypeptide of claim 48 , wherein the lyase activity comprises an ammonia lyase activity.
50 . The isolated or recombinant polypeptide of claim 49 , wherein the ammonia lyase activity comprises a phenylalanine ammonia lyase activity.
51 . The isolated or recombinant polypeptide of claim 49 , wherein the ammonia lyase activity comprises catalyzing the deamination of phenylalanine.
52 . The isolated or recombinant polypeptide of claim 49 , wherein the ammonia lyase activity comprises tyrosine ammonia lyase activity.
53 . The isolated or recombinant polypeptide of claim 49 , wherein the lyase activity comprises catalyzing the deamination of tyrosine.
54 . The isolated or recombinant polypeptide of claim 49 , wherein the ammonia lyase activity histidine ammonia lyase activity.
55 . The isolated or recombinant polypeptide of claim 54 , wherein the histidine ammonia lyase activity comprises generation of urocanoic acid or urocanoic acid derivatives.
56 . The isolated or recombinant polypeptide of claim 49 , wherein the lyase activity comprises catalyzing the deamination of histidine.
57 . The isolated or recombinant polypeptide of claim 48 , wherein the lyase activity comprises catalysis of the deamination of phenylalanine, tyrosine, histidine or derivatives thereof to generate cinnamic acid, para-hydroxycinnamic acid, para-hydroxyl styrene or derivatives thereof.
58 . The isolated or recombinant polypeptide of claim 48 , wherein the ammonia lyase activity comprises phenylalanine ammonia lyase, histidine ammonia lyase or tyrosine ammonia lyase activity or any combination thereof.
59 . The isolated or recombinant polypeptide of claim 48 , wherein the lyase activity comprises catalysis of the deamination of phenylalanine, tyrosine, histidine or derivatives thereof to generate L- or D-β-amino acids, or derivatives thereof.
60 . The isolated or recombinant polypeptide of claim 59 , wherein the lyase activity comprises catalysis of the deamination of histidine or derivatives thereof to generate L- and D-β-histidine, or derivatives thereof.
61 . The isolated or recombinant polypeptide of claim 48 , wherein the lyase activity comprises catalyzing the deamination of phenylalanine, tyrosine or histidine to generate a 3,5-dihydro-5-methylidine-4H-imidazol-4-one (MIO).
62 . The isolated or recombinant polypeptide of claim 48 , wherein the lyase activity comprises carboxy-lyase activity.
63 . The isolated or recombinant polypeptide of claim 48 , wherein the lyase activity comprises argininosuccinate lyase activity.
64 . The isolated or recombinant polypeptide of claim 48 , wherein the lyase activity comprises hydroxynitrile lyase activity.
65 . The isolated or recombinant polypeptide of claim 48 , wherein the lyase activity comprises C17,20-lyase activity.
66 . The isolated or recombinant polypeptide of claim 65 , wherein the lyase substrate is progesterone or 17-hydroxyprogesterone.
67 . The isolated or recombinant polypeptide of claim 48 , wherein the lyase activity comprises dimethylsulphoniopropionate (DMSP) lyase activity.
68 . The isolated or recombinant nucleic acid of claim 1 , wherein the lyase activity comprises 5′-deoxyribose phosphate lyase activity.
69 . The isolated or recombinant polypeptide of claim 48 , wherein the lyase activity comprises isocitrate lyase activity, catalyzing the formation of succinate and glyoxylate from isocitrate.
70 . The isolated or recombinant polypeptide of claim 69 , wherein the lyase activity comprises oxalomalate lyase activity, catalyzing the formation of oxaloacetate and glyoxylate.
71 . The isolated or recombinant polypeptide of claim 48 , wherein the lyase activity comprises hydroxymethylglutaryl-CoA lyase activity.
72 . The isolated or recombinant polypeptide of claim 48 , wherein the lyase activity is thermostable.
73 . The isolated or recombinant polypeptide of claim 72 , wherein the polypeptide retains a lyase activity under conditions comprising a temperature range of between about 37° C. to about 95° C., or between about 55° C. to about 85° C., or between about 70° C. to about 75° C., or between about 70° C. to about 95° C., or between about 90° C. to about 95° C., or retains a lyase activity in a temperature in the range between about 1° C. to about 5° C., between about 5° C. to about 15° C., between about 15° C. to about 25° C., between about 25° C. to about 37° C., or between about 37° C. to about 95° C., 96° C., 97° C., 98° C. or 99° C.
74 . The isolated or recombinant polypeptide of claim 48 , wherein the lyase activity is thermotolerant.
75 . The isolated or recombinant polypeptide of claim 74 , wherein the polypeptide retains a lyase activity after exposure to a temperature in the range from greater than 37° C. to about 95° C., from greater than 55° C. to about 85° C., or between about 70° C. to about 75° C., or from greater than 90° C. to about 95° C., or after exposure to a temperature in the range between about 1° C. to about 5° C., between about 5° C. to about 15° C., between about 15° C. to about 25° C., between about 25° C. to about 37° C., or between about 37° C. to about 95° C., 96° C., 97° C., 98° C. or 99° C.
76 . The isolated or recombinant polypeptide comprising a polypeptide as set forth in claim 48 and lacking a signal or leader sequence or a prepro sequence.
77 . An isolated or recombinant polypeptide comprising a polypeptide as set forth in claim 48 and having a heterologous signal or leader sequence or a heterologous prepro sequence.
78 . The isolated or recombinant polypeptide of claim 48 , wherein the lyase activity comprises a specific activity at about 37° C. in the range from about 100 to about 1000 units per milligram of protein, from about 500 to about 750 units per milligram of protein, from about 500 to about 1200 units per milligram of protein, or from about 750 to about 1000 units per milligram of protein.
79 . The isolated or recombinant polypeptide of claim 48 , wherein the thermotolerance comprises retention of at least half of the specific activity of the lyase at 37° C. after being heated to an elevated temperature, or, wherein the thermotolerance comprises retention of specific activity at 37° C. in the range from about 500 to about 1200 units per milligram of protein after being heated to an elevated temperature.
80 . The isolated or recombinant polypeptide of claim 48 , wherein the polypeptide comprises at least one glycosylation site, and optionally the glycosylation is an N-linked glycosylation, and optionally the polypeptide is glycosylated after being expressed in a yeast cell or mammalian cell, and optionally the yeast cell is P. pastoris or a S. pombe.
81 . The isolated or recombinant polypeptide of claim 48 , wherein the polypeptide retains a lyase activity under conditions comprising about pH 6.5, pH 6.0, pH 5.5, pH 5.0, pH 4.5 or 4.0 or more acidic, or after exposure to conditions comprising about pH 6.5, pH 6.0, pH 5.5, pH 5.0, pH 4.5 or 4.0 or more acidic.
82 . The isolated or recombinant polypeptide of claim 48 , wherein the polypeptide retains a lyase activity under conditions comprising about pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10 or pH 10.5 or more basic, or after exposure to conditions comprising about pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10 or pH 10.5 or more basic.
83 . A protein preparation comprising a polypeptide as set forth in claim 48 , wherein the protein preparation comprises a liquid, a solid or a gel.
84 . A heterodimer comprising a polypeptide as set forth in claim 48 and a second domain, wherein optionally the second domain is a polypeptide and the heterodimer is a fusion protein, and optionally the second domain comprises an epitope, an immunogenic peptide or a tag.
85 . A homodimer comprising a polypeptide as set forth in claim 48 .
86 . An immobilized polypeptide or an immobilized nucleic acid, wherein the polypeptide comprises a sequence as set forth in claim 48 , or a subsequence thereof, or the nucleic acid comprises a sequence as set forth in claim 1 , or a subsequence thereof, or the probe as set forth in claim 31 , wherein optionally the polypeptide or nucleic acid is immobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass, a micro electrode, a graphitic particle, a bead, a gel, a plate, an array or a capillary tube.
87 . An array comprising an immobilized polypeptide as set forth in claim 86 , or, an immobilized nucleic acid as set forth in claim 86 .
88 . An isolated or recombinant antibody that specifically binds to a polypeptide as set forth in claim 48 , wherein optionally the antibody is a monoclonal or a polyclonal antibody.
89 . A hybridoma comprising an antibody that specifically binds to a polypeptide as set forth in claim 48 .
90 . A method of isolating or identifying a polypeptide with a lyase activity comprising the steps of:
(a) providing an antibody as set forth in claim 88 ; (b) providing a sample comprising polypeptides; and (c) contacting the sample of step (b) with the antibody of step (a) under conditions wherein the antibody can specifically bind to the polypeptide, thereby isolating or identifying a polypeptide having a lyase activity.
91 . A method of making an anti-lyase antibody comprising
(a) administering to a non-human animal a nucleic acid as set forth in claim 1 or a subsequence thereof in an amount sufficient to generate a humoral immune response, thereby making an anti-lyase antibody, or (b) administering to a non-human animal a polypeptide as set forth in claim 48 or a subsequence thereof in an amount sufficient to generate a humoral immune response, thereby making an anti-lyase antibody.
92 . A method of producing a recombinant polypeptide comprising the steps of: (a) providing a nucleic acid operably linked to a promoter, wherein the nucleic acid comprises a sequence as set forth in claim 1 ; and (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide.
wherein optionally the method further comprises transforming a host cell with the nucleic acid of step (a) followed by expressing the nucleic acid of step (a), thereby producing a recombinant polypeptide in a transformed cell.
93 . A method for identifying a polypeptide having a lyase activity comprising the following steps:
(a) providing a polypeptide as set forth in claim 48 ; (b) providing a lyase substrate; and (c) contacting the polypeptide with the substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of a reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of the reaction product detects a polypeptide having a lyase activity.
94 . A method for identifying a lyase substrate comprising the following steps:
(a) providing a polypeptide as set forth in claim 48 ; (b) providing a test substrate; and (c) contacting the polypeptide of step (a) with the test substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of a reaction product identifies the test substrate as a lyase substrate.
95 . A method of determining whether a test compound specifically binds to a polypeptide comprising the following steps:
(a) expressing a nucleic acid or a vector comprising the nucleic acid under conditions permissive for translation of the nucleic acid to a polypeptide, wherein the nucleic acid has a sequence as set forth in claim 1 ; (b) providing a test compound; (c) contacting the polypeptide with the test compound; and (d) determining whether the test compound of step (b) specifically binds to the polypeptide.
96 . A method of determining whether a test compound specifically binds to a polypeptide comprising the following steps:
(a) providing a polypeptide as set forth in claim 48 ; (b) providing a test compound; (c) contacting the polypeptide with the test compound; and (d) determining whether the test compound of step (b) specifically binds to the polypeptide.
97 . A method for identifying a modulator of a lyase activity comprising the following steps:
(a) providing a polypeptide as set forth in claim 48 ; (b) providing a test compound; (c) contacting the polypeptide of step (a) with the test compound of step (b) and measuring an activity of the glucanase, wherein a change in the lyase activity measured in the presence of the test compound compared to the activity in the absence of the test compound provides a determination that the test compound modulates the lyase activity.
98 . The method of claim 97 , wherein the lyase activity is measured by providing a lyase substrate and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product, or, an increase in the amount of the substrate or a decrease in the amount of a reaction product,
wherein optionally a decrease in the amount of the substrate or an increase in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an activator of a lyase activity, and optionally an increase in the amount of the substrate or a decrease in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an inhibitor of a lyase activity.
99 . A computer system comprising a processor and a data storage device wherein said data storage device has stored thereon a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises sequence as set forth in claim 48 , a polypeptide encoded by a nucleic acid as set forth in claim 1 ,
wherein optionally the method further comprises a sequence comparison algorithm and a data storage device having at least one reference sequence stored thereon, or further comprises an identifier that identifies one or more features in said sequence and optionally the sequence comparison algorithm comprises a computer program that indicates polymorphisms.
100 . A computer readable medium having stored thereon a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 .
101 . A method for identifying a feature in a sequence comprising the steps of: (a) reading the sequence using a computer program which identifies one or more features in a sequence, wherein the sequence comprises a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises a polypeptide as set forth in claim 48 ; a polypeptide encoded by a nucleic acid as set forth in claim 1 ; and (b) identifying one or more features in the sequence with the computer program.
102 . A method for comparing a first sequence to a second sequence comprising the steps of: (a) reading the first sequence and the second sequence through use of a computer program which compares sequences, wherein the first sequence comprises a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises a polypeptide as set forth in claim 48 or a polypeptide encoded by a nucleic acid as set forth in claim 1 ; and (b) determining differences between the first sequence and the second sequence with the computer program.
wherein optionally the method further comprises a step of determining differences between the first sequence and the second sequence, or optionally the method further comprises the step of identifying polymorphisms, or optionally the method further comprises use of an identifier that identifies one or more features in a sequence, and optionally the method comprises reading the first sequence using a computer program and identifying one or more features in the sequence.
103 . A method for isolating or recovering a nucleic acid encoding a polypeptide with a lyase activity from a sample comprising the steps of:
(a) providing an amplification primer pair as set forth in claim 32 ; (b) isolating a nucleic acid from the sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to the amplification primer pair; and, (c) combining the nucleic acid of step (b) with the amplification primer pair of step (a) and amplifying nucleic acid from the sample, thereby isolating or recovering a nucleic acid encoding a polypeptide with a lyase activity from a sample, wherein optionally the sample is an environmental sample.
104 . A method for isolating or recovering a nucleic acid encoding a polypeptide with a lyase activity from a sample comprising the steps of:
(a) providing a polynucleotide probe comprising a sequence as set forth in claim 1 , or a subsequence thereof, or a probe as set forth in claim 31 ; (b) isolating a nucleic acid from the environmental sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to a polynucleotide probe of step (a); (c) combining the isolated nucleic acid or the treated environmental sample of step (b) with the polynucleotide probe of step (a); and (d) isolating a nucleic acid that specifically hybridizes with the polynucleotide probe of step (a), thereby isolating or recovering a nucleic acid encoding a polypeptide with a lyase activity from a sample, wherein optionally the sample is an environmental sample.
105 . The method of claim 103 or claim 104 , wherein the sample comprises an environmental sample, and optionally the environmental sample comprises a water sample, a liquid sample, a soil sample, an air sample or a biological sample, and optionally the biological sample is derived from a bacterial cell, a protozoan cell, an insect cell, a yeast cell, a plant cell, a fungal cell or a mammalian cell.
106 . A method of generating a variant of a nucleic acid encoding a polypeptide with a lyase activity comprising the steps of:
(a) providing a template nucleic acid comprising a sequence as set forth in claim 1 ; and (b) modifying, deleting or adding one or more nucleotides in the template sequence, or a combination thereof, to generate a variant of the template nucleic acid wherein optionally the method further comprises expressing the variant nucleic acid to generate a variant lyase polypeptide, and optionally the modifications, additions or deletions are introduced by a method comprising error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR), recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof and optionally the method is iteratively repeated until a lyase having an altered or different activity or an altered or different stability from that of a polypeptide encoded by the template nucleic acid is produced.
107 . The method of claim 106 , wherein the variant lyase polypeptide: (a) is thermotolerant, and retains some activity after being exposed to an elevated temperature; (b) has increased glycosylation as compared to the lyase-encoded by a template nucleic acid; or, (c) has a lyase activity under a high temperature, wherein the lyase-encoded by the template nucleic acid is not active under the high temperature.
108 . The method of claim 106 , wherein the method is iteratively repeated until (a) a lyase coding sequence having an altered codon usage from that of the template nucleic acid is produced, or, (b) a lyase gene having higher or lower level of message expression or stability from that of the template nucleic acid is produced.
109 . A method for modifying codons in a nucleic acid encoding a polypeptide with a lyase activity to increase its expression in a host cell, the method comprising the following steps:
(a) providing a nucleic acid encoding a polypeptide with a lyase activity comprising a sequence as set forth in claim 1 ; and, (b) identifying a non-preferred or a less preferred codon in the nucleic acid of step (a) and replacing it with a preferred or neutrally used codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to increase its expression in a host cell.
110 . A method for modifying codons in a nucleic acid encoding a lyase polypeptide, the method comprising the following steps:
(a) providing a nucleic acid encoding a polypeptide with a lyase activity comprising a sequence as set forth in claim 1 ; and, (b) identifying a codon in the nucleic acid of step (a) and replacing it with a different codon encoding the same amino acid as the replaced codon, thereby modifying codons in a nucleic acid encoding a lyase.
111 . A method for modifying codons in a nucleic acid encoding a lyase polypeptide to increase its expression in a host cell, the method comprising the following steps:
(a) providing a nucleic acid encoding a lyase polypeptide comprising a sequence as set forth in claim 1 ; and, (b) identifying a non-preferred or a less preferred codon in the nucleic acid of step (a) and replacing it with a preferred or neutrally used codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to increase its expression in a host cell.
112 . A method for modifying a codon in a nucleic acid encoding a polypeptide having a lyase activity to decrease its expression in a host cell, the method comprising the following steps:
(a) providing a nucleic acid encoding a lyase polypeptide comprising a sequence as set forth in claim 1 ; and (b) identifying at least one preferred codon in the nucleic acid of step (a) and replacing it with a non-preferred or less preferred codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in a host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to decrease its expression in a host cell, wherein optionally the host cell is a bacterial cell, a fungal cell, an insect cell, a yeast cell, a plant cell or a mammalian cell.
113 . A method for producing a library of nucleic acids encoding a plurality of modified lyase active sites or substrate binding sites, wherein the modified active sites or substrate binding sites are derived from a first nucleic acid comprising a sequence encoding a first active site or a first substrate binding site the method comprising the following steps:
(a) providing a first nucleic acid encoding a first active site or first substrate binding site, wherein the first nucleic acid sequence comprises a sequence that hybridizes under stringent conditions to a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:109, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249 or SEQ ID NO:251, or a subsequence thereof, and the nucleic acid encodes a lyase active site or a lyase substrate binding site; (b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and, (c) using the set of mutagenic oligonucleotides to generate a set of active site-encoding or substrate binding site-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of modified lyase active sites or substrate binding sites. wherein optionally a mutagenic oligonucleotide or a variant nucleic acid is generated by a method comprising an optimized directed evolution system, Gene Site-Saturation Mutagenesis (GSSM), or a synthetic ligation reassembly (SLR), error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof.
114 . A method for making a small molecule comprising the following steps:
(a) providing a plurality of biosynthetic enzymes capable of synthesizing or modifying a small molecule, wherein one of the enzymes comprises a lyase enzyme encoded by a nucleic acid comprising a sequence as set forth in claim 1 ; (b) providing a substrate for at least one of the enzymes of step (a); and (c) reacting the substrate of step (b) with the enzymes under conditions that facilitate a plurality of biocatalytic reactions to generate a small molecule by a series of biocatalytic reactions.
115 . A method for modifying a small molecule comprising the following steps:
(a) providing a lyase enzyme, wherein the enzyme comprises a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid comprising a nucleic acid sequence as set forth in claim 1 ; (b) providing a small molecule; and (c) reacting the enzyme of step (a) with the small molecule of step (b) under conditions that facilitate an enzymatic reaction catalyzed by the lyase enzyme, thereby modifying a small molecule by a lyase enzymatic reaction. wherein optionally step (b) comprises providing a plurality of small molecule substrates for the enzyme of step (a), thereby generating a library of modified small molecules produced by at least one enzymatic reaction catalyzed by the lyase enzyme; and optionally the method further comprises providing a plurality of additional enzymes under conditions that facilitate a plurality of biocatalytic reactions by the enzymes to form a library of modified small molecules produced by the plurality of enzymatic reactions; and optionally the method further comprises the step of testing the library to determine if a particular modified small molecule which exhibits a desired activity is present within the library, wherein optionally the step of testing the library further comprises the steps of systematically eliminating all but one of the biocatalytic reactions used to produce a portion of the plurality of the modified small molecules within the library by testing the portion of the modified small molecule for the presence or absence of the particular modified small molecule with a desired activity, and identifying at least one specific biocatalytic reaction that produces the particular modified small molecule of desired activity.
116 . A method for determining a functional fragment of a lyase enzyme comprising the steps of:
(a) providing a lyase enzyme, wherein the enzyme comprises a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 ; and (b) deleting a plurality of amino acid residues from the sequence of step (a) and testing the remaining subsequence for a lyase activity, thereby determining a functional fragment of a lyase enzyme. wherein optionally the lyase activity is measured by providing a lyase substrate and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product.
117 . A method for whole cell engineering of new or modified phenotypes by using real-time metabolic flux analysis, the method comprising the following steps:
(a) making a modified cell by modifying the genetic composition of a cell, wherein the genetic composition is modified by addition to the cell of a nucleic acid comprising a sequence as set forth in claim 1 ; (b) culturing the modified cell to generate a plurality of modified cells; (c) measuring at least one metabolic parameter of the cell by monitoring the cell culture of step (b) in real time; and, (d) analyzing the data of step (c) to determine if the measured parameter differs from a comparable measurement in an unmodified cell under similar conditions, thereby identifying an engineered phenotype in the cell using real-time metabolic flux analysis. wherein optionally the genetic composition of the cell is modified by a method comprising deletion of a sequence or modification of a sequence in the cell, or, knocking out the expression of a gene, and optionally the method further comprises selecting a cell comprising a newly engineered phenotype, and optionally the method further comprises culturing the selected cell, thereby generating a new cell strain comprising a newly engineered phenotype.
118 . An isolated or recombinant signal or leader sequence consisting of an amino acid sequence as set forth in the amino terminal residues 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44, 1 to 45, 1 to 46 or 1 to 47, of (a) an amino acid sequence as set forth in claim 48 ; or, (b) an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250 or SEQ ID NO:252.
119 . A chimeric polypeptide comprising at least a first domain comprising a signal peptide (SP) or leader sequence having an amino acid sequence as set forth in claim 118 , and at least a second domain comprising a heterologous polypeptide or peptide, wherein the heterologous polypeptide or peptide is not naturally associated with the signal peptide (SP) or leader sequence,
and optionally the heterologous polypeptide or peptide is not a lyase, and optionally the heterologous polypeptide or peptide is amino terminal to, carboxy terminal to or on both ends of the signal peptide (SP) or leader sequence.
120 . An isolated or recombinant nucleic acid encoding a chimeric polypeptide, wherein the chimeric polypeptide comprises at least a first domain comprising signal peptide (SP) or leader sequence having an amino acid sequence as set forth in claim 118 and at least a second domain comprising a heterologous polypeptide or peptide, wherein the heterologous polypeptide or peptide is not naturally associated with the signal peptide (SP) or leader sequence.
121 . An isolated or recombinant nucleic acid comprising a sequence encoding a polypeptide having a lyase activity and a signal sequence, wherein the nucleic acid comprises a sequence as set forth in claim 1 .
122 . The isolated or recombinant nucleic acid of claim 121 , wherein the signal sequence is derived from another lyase or a non-lyase enzyme.
123 . An isolated or recombinant nucleic acid comprising a sequence encoding a polypeptide having a lyase activity, wherein the sequence does not contain a signal sequence and the nucleic acid comprises a sequence as set forth in claim 1 .
124 . A method of increasing thermotolerance or thermostability of a lyase polypeptide, the method comprising glycosylating a lyase, wherein the polypeptide comprises at least thirty contiguous amino acids of a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 , thereby increasing the thermotolerance or thermostability of the lyase.
125 . A method for overexpressing a recombinant lyase in a cell comprising expressing a vector comprising a nucleic acid sequence as set forth in claim 1 , wherein overexpression is effected by use of a high activity promoter, a dicistronic vector or by gene amplification of the vector.
126 . A method of making a transgenic plant comprising the following steps:
(a) introducing a heterologous nucleic acid sequence into the cell, wherein the heterologous nucleic sequence comprises a sequence as set forth in claim 1 , thereby producing a transformed plant cell; (b) producing a transgenic plant from the transformed cell. wherein optionally the step (a) further comprises introducing the heterologous nucleic acid sequence by electroporation or microinjection of plant cell protoplasts, and optionally step (a) comprises introducing the heterologous nucleic acid sequence directly to plant tissue by DNA particle bombardment or by using an Agrobacterium tumefaciens host.
127 . A method of expressing a heterologous nucleic acid sequence in a plant cell comprising the following steps:
(a) transforming the plant cell with a heterologous nucleic acid sequence operably linked to a promoter, wherein the heterologous nucleic sequence comprises a sequence as set forth in claim 1 ; (b) growing the plant under conditions wherein the heterologous nucleic acids sequence is expressed in the plant cell.
128 . A method for deaminating a phenylalanine, tyrosine or a histidine comprising the following steps:
(a) providing a polypeptide having a lyase activity as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 ; (b) providing a composition comprising a phenylalanine, tyrosine or a histidine residue; and (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the lyase deaminates the phenylalanine, tyrosine or a histidine residue in the composition. wherein optionally the composition comprises a plant cell, a bacterial cell, a yeast cell, an insect cell, or an animal cell, and optionally the polypeptide has ammonia lyase activity.
129 . A dough or a bread product comprising a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 , wherein optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity.
130 . A method of dough conditioning comprising contacting a dough or a bread product with at least one polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 , under conditions sufficient for conditioning the dough.
131 . A beverage or drink comprising a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 , wherein optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity.
A method of beverage production comprising administration of at least one polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 , to a beverage, drink or a beverage or drink precursor under conditions sufficient for decreasing the viscosity of the beverage.
132 . A food, a feed or a nutritional supplement comprising a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 , wherein optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity.
133 . A method for utilizing a lyase as a nutritional supplement in an animal diet, the method comprising:
preparing a nutritional supplement containing a lyase enzyme comprising at least thirty contiguous amino acids of a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 ; and administering the nutritional supplement to an animal as an enzyme substitution therapy, wherein optionally the enzyme substitution therapy is for the treatment of phenylketonuria (PKU), and optionally the animal is a human, a ruminant or a monogastric animal, and optionally the lyase enzyme is prepared by expression of a polynucleotide encoding the lyase in an organism selected from the group consisting of a bacterium, a yeast, a plant, an insect, a fungus and an animal, and optionally the organism is selected from the group consisting of an S. pombe, S. cerevisiae, Pichia pastoris, E. coli, Streptomyces sp., Bacillus sp. and Lactobacillus sp.
134 . An edible enzyme delivery matrix or pellet comprising a thermostable recombinant lyase enzyme comprising a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 , wherein optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity.
135 . A method for delivering a lyase enzyme supplement to an animal, the method comprising: preparing an edible enzyme delivery matrix or pellets comprising a granulate edible carrier and a thermostable recombinant lyase enzyme, wherein the pellets readily disperse the lyase enzyme contained therein into aqueous media, and the recombinant lyase enzyme comprises a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 ; and, administering the edible enzyme delivery matrix or pellet to the animal,
and optionally the lyase enzyme is glycosylated to provide thermostability at pelletizing conditions.
136 . A kit comprising a polypeptide as set forth in claim 48 , or a nucleic acid as set forth in claim 1 , or an antibody as set forth in claim 88 , wherein optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity.
137 . A wood, wood pulp or wood product comprising a lyase as set forth in claim 48 , or a lyase encoded by a nucleic acid as set forth in claim 1 , wherein optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity.
138 . A paper, paper pulp or paper product comprising a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 , wherein optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity.
139 . A detergent composition comprising a lyase as set forth in claim 48 , or a lyase encoded by a nucleic acid as set forth in claim 1 ,
wherein optionally the polypeptide is formulated in a non-aqueous liquid composition, a cast solid, a granular form, a particulate form, a compressed tablet, a gel form, a paste or a slurry form, and optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity.
140 . A pharmaceutical composition or dietary supplement comprising a lyase as set forth in claim 48 , or a lyase encoded by a nucleic acid as set forth in claim 1 ,
wherein optionally the lyase is formulated as a tablet, gel, geltab, pill, implant, liquid, spray, powder, food, feed pellet, as an injectable formulation or as an encapsulated formulation, and optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity.
141 . The pharmaceutical composition or dietary supplement of claim 140 , formulated for the treatment of phenylketonuria (PKU).
142 . The pharmaceutical composition or dietary supplement of claim 140 , wherein the polypeptide is chemically modified.
143 . The pharmaceutical composition or dietary supplement of claim 142 , wherein the polypeptide is chemically modified to produce a protected form that possesses better specific activity, prolonged half-life, and/or reduced immunogenicity in vivo.
144 . The pharmaceutical composition or dietary supplement of claim 140 , wherein the polypeptide is chemically modified by glycosylation, pegylation or a combination thereof.
145 . The pharmaceutical composition or dietary supplement of claim 140 , wherein the polypeptide is formulated by encapsulation in a liposome, or a micro- or nano-structure, wherein optionally the structure is a nanotubule or a nano- or microcapsule.
146 . The pharmaceutical composition or dietary supplement of claim 140 , wherein the polypeptide is formulated in a matrix stabilized enzyme crystal.
147 . A method for decreasing elevated levels of phenylalanine (Phe) in the bloodstream (hyperphenylalaninemia) comprising the following steps:
(a) providing a pharmaceutical composition or dietary supplement of any of claims 140 to 143 ; and, (b) administering an effective amount of the pharmaceutical composition or dietary supplement to an individual in need thereof.
148 . A method for processing a biomass material comprising contacting a composition with a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 ,
wherein optionally the biomass material is derived from an agricultural crop, is a byproduct of a food or a feed production, is a lignocellulosic waste product, or is a plant residue or a waste paper or waste paper product, and optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity, and optionally the plant residue comprise stems, leaves, hulls, husks, corn cobs, corn stover, straw, wood, wood chips, wood pulp and sawdust, and optionally the paper waste comprises discarded or used photocopy paper, computer printer paper, notebook paper, notepad paper, typewriter paper, newspapers, magazines, cardboard and paper-based packaging materials, and optionally the processing of the biomass material generates a bioethanol.
149 . A dairy product comprising a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 , wherein optionally the dairy product comprises a milk, an ice cream, a cheese or a yoghurt, and optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity.
150 . A method for improving texture and flavor of a dairy product comprising the following steps: (a) providing a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 ; (b) providing a dairy product; and (c) contacting the polypeptide of step (a) and the dairy product of step (b) under conditions wherein the lyase can improve the texture or flavor of the dairy product.
151 . A textile or fabric comprising a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 , wherein optionally the textile or fabric comprises a cellulose-containing fiber, and optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity.
152 . A method for treating solid or liquid animal waste products comprising the following steps:
(a) providing a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 , wherein optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity; (b) providing a solid or a liquid animal waste; and (c) contacting the polypeptide of step (a) and the solid or liquid waste of step (b) under conditions wherein the protease can treat the waste.
153 . A processed waste product comprising a polypeptide as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 , wherein optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity.
154 . A disinfectant comprising a polypeptide having a lyase activity, wherein the polypeptide comprises a sequence as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 , wherein optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity.
155 . A biodefense or bio-detoxifying agent comprising a polypeptide having a lyase activity, wherein the polypeptide comprises a sequence as set forth in claim 48 , or a polypeptide encoded by a nucleic acid as set forth in claim 1 , wherein optionally the polypeptide has ammonia lyase activity, or phenylalanine ammonia lyase activity, tyrosine ammonia lyase activity and/or histidine ammonia lyase activity.
156 . A method for processing a biomass material comprising providing a mixture of enzymes, wherein at least one of the mixture of enzymes is a polypeptide of claim 48 having lyase activity, and contacting the enzyme mixture with the biomass material, wherein optionally the biomass material is derived from an agricultural crop, is a byproduct of a food or a feed production, or is a plant residue or a waste paper or waste paper product, and optionally at least one of the mixture of enzymes has activity comprising a cellulase, endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase activity, and optionally the plant residue comprise stems, leaves, hulls, husks, corn cobs, corn stover, straw, wood, wood chips, wood pulp and sawdust, and optionally the paper waste comprises discarded or used photocopy paper, computer printer paper, notebook paper, notepad paper, typewriter paper, newspapers, magazines, cardboard and paper-based packaging materials.Join the waitlist — get patent alerts
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