Transferases and oxidoreductases, nucleic acids encoding them and methods for making and using them
View Patent ↗This invention relates generally to enzymes, polynucleotides encoding the enzymes, the use of such polynucleotides and polypeptides and more specifically to enzymes having transferase activity, e.g., transaminase activity, e.g., d-amino-acid transferase activity, and/or oxidoreductase activity, e.g., dehydrogenase activity, e.g., damino-acid dehydrogenase activity, and/or catalyze the transfer of a chemical group, catalyze transamination, catalyze the reaction: D-alanine+2-oxoglutarate<=>pyruvate+D-glutamate, and/or catalyze an oxidation-reduction reaction, catalyze the removal of hydrogen atoms, and/or catalyze the reaction: D-amino acid+H 2 O+acceptor<=>a 2-oxo acid+NH 3 +reduced acceptor. Thus, the invention provides enzymes, compositions, methods for production of pharmaceutical compositions, pharmaceutical intermediates, antibiotics, sweeteners, peptide enzymes, peptide hormones, fuel and fuel additive compositions, foods and food additives, beverage and beverage additives, feeds and feed additives, drugs and drug additives, dietary supplements, textiles, wood, paper, pulp, and detergents comprising the polypeptides or polynucleotides in accordance with the invention.
1. An isolated, synthetic, or recombinant polypeptide having a d-amino-acid transferase activity, wherein the polypeptide is the amino acid sequence of SEQ ID NO:220 or the polypeptide is selected from a group consisting of:
(a) a polypeptide variant having d-amino-acid transferase activity wherein the polypeptide variant has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the full length of the amino acid sequence of SEQ ID NO:220;
(b) a polypeptide having d-amino-acid transferase activity and the amino acid sequence encoded by SEQ ID NO:219
(c) a polypeptide having d-amino-acid transferase activity wherein the polypeptide is a variant of the amino acid sequence of SEQ ID NO:220 and at least one mutation of SEQ ID NO:220 selected from the group consisting of: D2A, D2D, D2F, D2G, D2H, D2I, D2N, D2Q, D2R, D2T, D2V, D2Y, Y6C, Y6F, Y6H, Y6L, Y6M, G9A, N10R, N10S, N10T, N10W, G12A, L14L, L14V, T18W, V19T, P20S, N40N, G41G, G41Y, V42M, V42V, A45L, L46A, L46H, D47W, N55L, S56S, A57M, E61D, E61R, E61Y, 162C, 164H, L66G, L66L, E69Y, K72M, K73L, T74A, T74L, T74M, T74R, T74V, N76C, E77R, Y79R, Y79V, S80L, S80R, V82A, G85D, G85G, V93A, V93G, V93L, V93Y, G98A, S101Q, S101S, V103T, A106G, A106W, P108C, P108F, P108T, N110A, P117W, N118A, N118G, N118K, N118R, I120L, K126K, K126Q, K126R, I127M, K128L, K128R, K128S, L129I, L129V, I130M, T131T, M132A, M132R, M132Y, D133E, D133L, D133N, F137W, L138L, P149P, N150S, I152V, R156A, R156W, G161G, H163K, H163R, A165T, H168A, E171R, E171S, T172I, C176G, A177S, H182G, K185R, K185T, N186S, S205A, Q209S, V212E, R213W, I216T, P217H, P217V, D219F, E220V, R221E, F223C, S226P, L228F, V234A, V236T, S238S, G240G, T241C, T241R, L242F, E248F, D250E, G256K, K257V, E260G, L262R, K263M, I265L, D267G, D267R, E268S, L270G, L270S, L270W, R271S, I274W, G278S, Y279C, T280N, P281W, E282G, W283A, W283E, W283G, W283R, W283S, W283T, W283V, S284R, R285F, V286G, and V286R; or,
(d) a polypeptide having d-amino-acid transferase activity wherein the polypeptide is a variant of the amino acid sequence of SEQ ID NO:220 and a combination of mutations of SEQ ID NO:220 selected from the group consisting of:
Number/
designation
Mutation
1
P20S-N118G
2
T74V-V93G-L270W
3
P20S-T74V-L270W
4
T74V-L270W
5
P20S-K73L-T241R-L270W
6
K73L-V93G-V236T-T241R
7
P20S-T74V-V236T
8
P20S-K73L-V93G
9
K73L-V236T
10
P20S-L270W
11
2N-P20S-K73L-V93G-N118G
12
P20S-T74V-N118A
13
P20S-V236T
14
P20S-T241R-L270W
15
P20S-T241R
16
T74V-V93G-V236T-T241R
17
P20S-K73L-T74V-L270W
18
P20S-V93G-V236T
19
P20S-K73L-T74V-T241R-L270W
20
P20S-K73L-L270W
21
T74V-V93G-N118G-V236T-T241R
22
P20S-K73L-210A (SILENT-GCC → GCT)-V236T
23
N118A-L270W
24
P20S-58K (SILENT AAG → AAA)-L270W
25
P20S-V93G-N118G
26
P20S-V236T-T241R
27
P205-P117W-N118A-V236T-L270W
28
V93G-V236T
29
V236T-L270W
30
P20S-N118G-L270W
30
P20S-N110A-N118G
31
N110A-N118G-T241R-L270W
32
P20S-T74V-V93G-N110A-N118G-V236T-L270W
33
V93G-N110A-N118G-V236T
34
P20S-T74V-N110A-N118G
35
N110A-N118G
36
P20S-V93G-N110A-N118G-T241R
37
N110A-N118A-L270W
38
P20S-N110A-N118G-L270W
39
N110A-N118A-V236T-T241R
40
N110A-N118G-L270W
41
V93G-N110A-N118G-T241R
42
P20S-V93G-N110A-N118G
43
V93G-N110A
44
P20S-N110A-N118G-V236T
45
T74V-N110A-N118A-V236T
46
P20S-K73L-T74V-N110A-N118G-V236T-T241R
47
86E (SILENT GAG → GAA)-N110A-N118A-V236T
48
T74V-N118G
49
P20S-T241R-L270W-277T
(SILENT ACA → ACG)
50
T74V-N118A-L270W
51
P20S-K73L-N118A-L270W
52
P20S-V93G-T241R
53
T74V-V93G-N110A-T241R
54
V93G-N110A-N118A
55
P20S-T74V-V93G-N110A-N118G-T241R
56
T74V-N110A-N118G-L270W
57
P205-231A (SILENT GCG → GCA)-V236T
58
V93G-V236T-T241R.
2. A protein preparation comprising the polypeptide of claim 1 , wherein the protein preparation comprises a liquid, a solid or a gel.
3. An immobilized polypeptide comprising the polypeptide of claim 1 , wherein optionally the polypeptide is immobilized on a wood chip, a paper, a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an array or a capillary tube.
4. An enzyme mixture or cocktail comprising
(a) the polypeptide of claim 1 , and one or more other enzyme(s);
(b) the mixture or cocktail of (a), wherein the one or more other enzyme(s) is another transferase, e.g., transaminase, e.g., d-amino-acid transferase, and/or oxidoreductase, e.g., dehydrogenase, e.g., d-amino-acid dehydrogenase, a mannanase and/or a glucanase, cellulases, lipases, esterases, proteases, or endoglycosidases, endo-beta.-1,4-glucanases, beta-glucanases, endo-beta-1,3(4)-glucanases, cutinases, peroxidases, catalases, laccases, amylases, glucoamylases, pectinases, racemases, epimerases, isomerases, oxidorecuctases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases, mannanases, xyloglucanases, xylanase, a mannanase and/or a glucanases, pectin acetyl esterases, rhamnogalacturonan acetyl esterases, polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases, pectin methylesterases, cellobiohydrolases and/or transglutaminases.
5. A beverage or beverage precursor comprising the polypeptide of claim 1 , or the enzyme mixture or cocktail of claim 4 .
6. A method of beverage production comprising administration of the polypeptide of claim 1 , or the enzyme mixture or cocktail of claim 4 , to a beverage or a beverage precursor, wherein optionally the beverage or beverage precursor is a wort or a beer.
7. A feed, food, food or feed additive, food or feed supplement, or dietary aid or diet supplement comprising the polypeptide of claim 1 , or the enzyme mixture or cocktail of claim 4 , wherein optionally the feed, food, food or feed additive, food or feed supplement, or dietary aid further comprises a carrier selected from the group consisting of a grain germ, a grain germ that is spent of oil, a hay, an alfalfa, a timothy, a soy hull, a sunflower seed meal and a wheat midd, and optionally the carrier comprises grain germ that is spent of oil, or optionally the transferase, e.g., the transaminase, e.g., the d-amino-acid transferase, and/or the oxidoreductase, e.g., the dehydrogenase, e.g., the d-amino-acid dehydrogenase enzyme is glycosylated to provide thermostability at pelletizing conditions, and optionally the delivery matrix is formed by pelletizing a mixture comprising a grain germ and a transferase, e.g., a transaminase, e.g., a d-amino-acid transferase, and/or an oxidoreductase, e.g., a dehydrogenase, e.g., a d-amino-acid dehydrogenase, and optionally the pelletizing conditions include application of steam, and optionally the pelletizing conditions comprise application of a temperature in excess of about 80° C. for about 5 minutes and the enzyme retains a specific activity of at least 350 to about 900 units per milligram of enzyme.
8. A chewing gum, lozenge or candy comprising the polypeptide of claim 1 , or the enzyme mixture or cocktail of claim 4 .
9. A chimeric transferase, e.g., transaminase, e.g., d-amino-acid transferase, and/or oxidoreductase, e.g., dehydrogenase, e.g., d-amino-acid dehydrogenase comprising the polypeptide of claim 1 and at least one heterologous binding domain; wherein optionally the binding domain comprises: a NAD, a NAD(P), a calcium, a thiamine, a FAD, a zinc, a DNA and/or a lipoyl binding domain.
10. A dough, bread or baked products and/or dough, bread or baked product precursors comprising the polypeptide of claim 1 or the enzyme mixture or cocktail of claim 4 .
11. A method of making a pyruvate and/or a D-glutamate comprising:
(a) providing a D-alanine and a 2-oxoglutarate;
(b) providing the polypeptide of claim 1 ; and
(c) contacting the polypeptide of (b) with the D-alanine+2-oxoglutarate under conditions wherein the polypeptide catalyzes the reaction D-alanine+2-oxoglutarate<=>pyruvate+D-glutamate.
12. A method of making a 2-oxo acid comprising
(i) (a) providing a D-amino acid+H 2 O+acceptor;
(b) providing the transaminase polypeptide of claim 1 ; and
(c) contacting the polypeptide of (b) with the D-amino acid+H 2 O+acceptor, under conditions wherein the polypeptide catalyzes the reaction: D-amino acid+H 2 O+acceptor<=>a 2-oxo acid+NH3+reduced acceptor; or
(ii) the method of (i), wherein the acceptor is a benzoquinone is
(iii) the method of (ii) wherein the benzoquinone is a 1,2-benzoquinone or a 1,4-benzoquinone, or ubiquinone, ubidecarenone or coenzyme Q.
13. A method for transferring an amino group from an amino acid to an alpha-keto acid comprising
(i) (a) providing an amino acid;
(b) providing the transaminase polypeptide of claim 1 ; and
(c) contacting the polypeptide of (b) with the amino acid under conditions wherein the polypeptide catalyzes the conversion of the amino acid to an alpha-keto acid; or
(ii) the method of (i), wherein the transaminase activity comprises catalyzing the conversion of a racemic amino acid mixture to a substantially optically pure alpha-keto acid.
14. A method for transferring making an amino acid from an alpha-keto acid comprising
(i) (a) providing an alpha-keto acid
(b) providing the transaminase polypeptide of claim 1 ; and
(c) contacting the polypeptide of (b) with the alpha-keto acid under conditions wherein the polypeptide catalyzes the conversion of the alpha-keto acid to an amino acid;
(ii) the method of (i), wherein the transaminase activity comprises catalyzing the conversion of a racemic alpha-keto mixture to a substantially optically pure D- or L-amino acid; or
(iii) the method of (i) or (ii), wherein oxaloacetate is converted to an aspartate, or α-ketoglutarate is converted to glutarate, or an α-ketoisovalerate is converted to an L-valine; or the transaminase activity is an omega transaminase activity that catalyzes the conversion of isobutylamine to isobutyraldehyde.
15. A method of catalyzing the conversion of an amine to a ketone comprising
(i) (a) providing an amine;
(b) providing the transaminase polypeptide of claim 1 ; and
(c) contacting the polypeptide of (b) with the amine under conditions wherein the polypeptide catalyzes the conversion of an amine to a ketone, wherein the amine is not in or from a tryptophan (with the proviso that the second amino acid is not tryptophan, or with the proviso that the amine is not in or from a tryptophan);
(ii) the method of (i), wherein the transaminase activity comprises catalyzing the conversion of a chiral amine to a ketone; or
(iii) the method of (i) or (ii), wherein the amine is a ω-amine.
16. A method of catalyzing the synthesis of an amino acid comprising
(i) (a) providing amino acid and a keto acid, wherein the amino acid is not tryptophan;
(b) providing the transaminase polypeptide of claim 1 ; and
(c) contacting the polypeptide of (b) with the amino acid and keto acid under conditions wherein a second amino acid and a pyruvate is produced, wherein the second amino acid is not tryptophan (with the proviso that the second amino acid is not tryptophan); or
(ii) the method of (i), further comprising reacting the pyruvate with an acetolactate synthase enzyme under conditions appropriate to produce a compound that does not react with transaminase enzyme;
(iii) the method of (ii), wherein the compound that does not react with transaminase enzyme is acetolactate or acetoin; or, the first amino acid is alanine or L-aspartate; or, the keto acid is 2-ketobutyrate or tri-methyl pyruvate; or, the second amino acid is 2-aminobutyrate or tert-leucine.
17. An isolated, synthetic, or recombinant polypeptide having a d-amino-acid transferase activity comprising SEQ ID NO:220.
18. The polypeptide of claim 1 , further comprising at least one amino acid residue substitution, and the polypeptide retains d-amino-acid transferase activity.
19. The polypeptide of claim 18 , wherein the amino acid 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.
20. The polypeptide of claim 19 , wherein 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.
21. The polypeptide of claim 17 , wherein the amino acid sequence is lacking a signal sequence, a prepro domain, or a binding domain.
22. The polypeptide of claim 21 , wherein the binding domain comprises, or consists of, a NAD, a NAD(P), a calcium, a thiamine, a FAD, a zinc, a DNA and/or a lipoyl binding domain.
23. The polypeptide of claim 17 , wherein the amino acid sequence further comprising a heterologous sequence.
24. The polypeptide of claim 23 , wherein the heterologous sequence consists of: (A) a heterologous signal sequence, a heterologous domain, a heterologous binding domain, a heterologous dockerin domain, a heterologous catalytic domain (CD), or a combination thereof; (B) the sequence of (A), wherein the heterologous signal sequence, binding domain or catalytic domain (CD) is derived from a heterologous enzyme; and/or, (C) a tag, an epitope, a targeting peptide, a cleavable sequence, a detectable moiety or an enzyme.
25. The polypeptide of claim 24 , wherein the heterologous sequence or the heterologous binding domain comprises: a NAD, a NAD(P), a calcium, a thiamine, a FAD, a zinc, a DNA and/or a lipoyl binding domain.
26. An isolated, synthetic, or recombinant polypeptide having the amino acid sequence of SEQ ID NO:220, and having d-amino acid transferase activity and an enzymatically active fragments thereof.