US2017143004A1PendingUtilityA1
Buttiauxella sp. phytase variants
Est. expiryApr 18, 2028(~1.7 yrs left)· nominal 20-yr term from priority
Inventors:Oliver KenschKlaus PellengahrBirgitta LeuthnerJayarama K. ShettyMichael J. PepsinSoren DalsgaardMai Faurschou Isaksen
C12P 19/02C12P 19/14A23K 20/189C12P 7/06A23L 29/06C12N 9/16A23V 2002/00C12Y 301/03026Y02E50/10
60
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Claims
Abstract
Provided herein are variants of Buttiauxella sp. phytases that may be used in industrial applications including methods for starch liquefaction, alcohol fermentations and for enhancing phosphate digestion in foods and animal feeds.
Claims
exact text as granted — not AI-modified1 . A phytase variant having improved thermal activity of at least about 5° C. as compared to wild type Buttiauxella sp. phytase (SEQ ID NO: 1).
2 . The phytase variant of claim 1 , wherein the improved thermal activity is at least about 17° C. as compared to the phytase SEQ ID NO: 1.
3 . The phytase variant of claim 1 or claim 2 , wherein the improved thermal activity is in a range from about 17 to about 21° C.
4 . The phytase variant of any one of claims 1 to 3 , wherein the improved thermal activity is in a range from about 17.4 to about 20.2° C.
5 . A thermostable phytase variant capable of retaining greater than 50% of its activity after exposure to an elevated temperature for 10 minutes at pH 5.5 in buffer, wherein the elevated temperature is at least about 5° C. higher than a temperature at which wild type Buttiauxella sp. phytase (SEQ ID NO: 1) retains greater than 50% of its activity.
6 . The thermostable phytase variant of claim 5 , wherein the elevated temperature is at least about 20.5° C. higher.
7 . The thermostable phytase variant of claim 5 or claim 6 , wherein the elevated temperature is in a range from about 20.5° C. to about 27° C.
8 . The thermostable phytase variant of any one of claims 5 to 7 , wherein the elevated temperature is in a range from about 20.2° C. to about 26.8° C.
9 . A phytase variant having phytase activity and an amino acid sequence that varies from the amino acid sequence of wild type Buttiauxella sp. phytase (SEQ ID NO: 1), wherein the amino acid sequence of the phytase variant comprises a variation at one or more positions corresponding to position 75, 76, 77, 198, 367 or 374 of SEQ ID NO: 1.
10 . The phytase variant of claim 9 , wherein the phytase variant further comprises one or more additional variation, wherein the variation is 92A, 164E/S, 1711/V, 192A or
11 . The phytase variant of claim 9 or claim 10 wherein the variation at one or more of positions 75, 76, 77, 198, 367 or 374 respectively is 75P, 76R, 77S, 198R, 367L or 374P and the phytase variant further comprises one or more additional variation wherein the variation is 92A, 164E/S, 171V, 192A or 256A/E/P.
12 . The phytase variant of any of claims 9 to 11 , wherein the phytase variant further comprises one or more additional variation, wherein the one or more additional variation position is 26, 37, 89, 134, 160, 176, 178, 188, 190, 207, 209, 211, 235, 261, 270, 303 or 318.
13 . The phytase variant of claim 12 , wherein the one or more additional variation at position 26, 37, 89, 134, 160, 176, 178, 188, 190, 207, 209, 211, 235, 261, 270, 303 or 318 is respectively 26E, 037Y, 089T, 1341/V, 160R, 176K, 178P, 188N, 190E, 207E/T, 209S, 211C, 235V, 261E, 270K, 303F or 318D.
14 . The phytase variant of any of claims 9 to 13 , wherein the phytase variant further comprises one or more additional variation, wherein the one or more additional variation position is 1, 10, 11, 38, 66, 71, 81, 92, 109, 111, 119, 120, 121, 141, 142, 152, 155, 193, 214, 239, 245, 248, 255, 268, 277, 283, 285, 287, 288, 293, 296, 314, 337, 345, 350, 364, 371, 372, 396, 399, 406 or 413.
15 . The phytase variant of claim 14 , wherein the one or more additional variation at position 1, 10, 11, 38, 66, 71, 81, 92, 109, 111, 119, 120, 121, 141, 142, 152, 155, 193, 214, 239, 245, 248, 255, 268, 277, 283, 285, 287, 288, 293, 296, 314, 337, 345, 350, 364, 371, 372, 396, 399, 406 or 413 is respectively 1S, 10I, 11I, 38S, 66E, 71K, 81A, 92E, 109Q, 111G, 119N, 120L, 121E, 141R, 142L, 152M, 155E, 193Q, 211C, 214V, 235V, 239K, 245D, 248L, 255A, 261E, 268A/T, 270K, 277T, 283D, 285K, 287D, 288A, 293G or 296S.
16 . A phytase variant having phytase activity and an amino acid sequence that varies from the amino acid sequence of wild type Buttiauxella sp. phytase (SEQ ID NO: 1), wherein the amino acid sequence of the phytase variant comprises at least one variation as compared with SEQ ID NO: 1, and wherein the phytase sequence variation comprises
a) N37Y, 575P, A89T, D92A, T1341, H160R, F164E, T171V, T176K, A178P, S188P, G192A, K198R, K207E, A2095, S248L, Q256Y, A261E, N270K, A374P b) N37Y, G77S, A89T, D92A, T1341, H160R, F164E, T171V, T176K, A178P, S188P, G192A, K198R, K207E, A2095, S248L, Q256Y, A261E, N270K, A374P c) N37Y, 575P, Q76R, A89T, D92A, T1341, H160R, F164E, T1711, T176K, A178P, S188P, G192A, K207E, A2095, A235V, S248L, Q256Y, A261E, N270K, A374P d) N37Y, A89T, D92A, T1341, F164E, T171V, T176K, A178P, G192A, K207E, A2095, A235V, S248L, Q256P, A261E, N270K, A374P e) 575P, Q76R, A89T, D92A, T1341, H160R, F164E, T1711, T176K, A178P, S188P, G192A, K207E, A2095, S248L, Q256Y, A261E, N270K, A374P f) N37Y, Q76R, A89T, D92A, T1341, H160R, F164E, T1711, T176K, A178P, S188P, G192A, K207E, A2095, S248L, Q256Y, A261E, N270K, A374P g) N37Y, Q76R, A89T, D92A, T1341, F1645, T171V, T176K, A178P, S188P, G192A, K207E, A2095, A235V, S248L, Q256A, A261E, N270K, A374P h) 575P, A89T, D92A, T1341, F164E, T171V, T176K, A178P, S188P, G192A, K207E, A2095, A235V, S248L, Q256Y, A261E, N270K, A374P i) 575P, Q76R, A89T, D92A, T1341, H160R, F164E, T171V, T176K, A178P, S188P, G192A, K207E, A2095, A235V, S248L, Q256Y, A261E, N270K, P367L, A374P j) N37Y, A89T, D92A, T1341, F164E, T1711, T176K, A178P, G192A, K207E, A209S, A235V, S248L, Q256Y, A261E, N270K, A374P k) N37Y, Q76R, A89T, D92A, T1341, F164E, T171V, T176K, A178P, G192A, K207E, A209S, S248L, Q256Y, A261E, N270K, A374P l) N37Y, Q76R, A89T, D92A, T1341, F164E, T171V, T176K, A178P, G192A, K207E, A209S, S248L, Q256A, A261E, N270K, A374P m) N37Y, S75P, Q76R, A89T, D92A, T1341, F164E, T171V, T176K, A178P, K207E, A209S, A235V, S248L, Q256A, A261E, N270K, A374P n) N37Y, S75P, A89T, D92A, T1341, H160R, F164E, T171V, T176K, A178P, K207E, A209S, A235V, S248L, Q256Y, A261E, N270K, A374P o) N37Y, A89T, D92A, T1341, H160R, F164S, T1711, T176K, A178P, S188P, G192A, K207E, A209S, A235V, S248L, Q256E, A261E, N270K, A374P p) A89T, D92A, T1341, H160R, F164E, T171V, T176K, A178P, G192A, K207E, A209S, A235V, S248L, Q256Y, A261E, N270K, A374P q) N37Y, S75P, A89T, D92A, T1341, H160R, F164S, T171V, T176K, A178P, S188P, K207E, A209S, S248L, Q256H, A261E, N270K, A374P r) N37Y, S75P, A89T, D92A, T1341, F164S, T171V, T176K, A178P, S188P, G192A, K207E, A209S, S248L, Q256A, A261E, N270K, A374P s) S75P, Q76R, A89T, D92A, T1341, H160R, F164E, T171V, T176K, A178P, G192A, K207E, A209S, S248L, Q256A, A261E, N270K, A374P; or t) N37Y, Q76R, A89T, D92A, T1341, H160R, F164S, T171V, T176K, A178P, G192A, K207E, A209S, A235V, S248L, Q256Y, A261E, N270K, A374P.
17 . The phytase variant of claim 16 , wherein the phytase sequence variation comprises N37Y, S75P, Q76R, A89T, D92A, T134I, H160R, F164E, T171I, T176K, A178P, S188P, G192A, K207E, A209S, A235V, S248L, Q256Y, A261E, N270K, A374P.
18 . A phytase which has at least a minimum percent sequence identity and/or percent homology to the phytase of claims 1 to 17 , wherein the minimum percent identity and/or homology is at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.
19 . A phytase variant having phytase activity and an amino acid sequence that varies from the amino acid sequence of the wild type Buttiauxella sp. phytase (SEQ ID NO: 1), wherein the amino acid sequence of the phytase variant comprises at least one variation as compared with SEQ ID NO: 1, and wherein the variant has a variation at at least any one of the following positions: 70, 193, 197, 221 and 407.
20 . A phytase of claim 19 , wherein the variant has at least the following variations: N70Y, H193R, F197E, S221P and A407P.
21 . A phytase variant according to any one of claims 1 to 4 wherein said phytase comprises any one or more of the features of claims 5 to 8 and/or claims 9 to 15 and/or claim 16 or 17 and/or claim 18 and/or claim 19 or 20 .
22 . A phytase variant according to any one of claims 5 to 8 wherein said phytase comprises any one or more of the features of claims 1 to 4 and/or claims 9 to 15 and/or claim 16 or 17 and/or claim 18 and/or claim 19 or 20 .
23 . A phytase variant according to any one of claims 9 to 15 wherein said phytase comprises any one or more of the features of claims 1 to 4 , and/or claims 5 to 8 and/or claim 16 or 17 and/or claim 18 and/or claim 19 or 20 .
24 . A phytase variant according to any one of claims 16 to 17 wherein said phytase comprises any one or more of the features of claims 1 to 4 and/or claims 5 to 8 and/or claims 9 to 15 and/or claim 18 and/or claim 19 or 20 .
25 . A phytase variant according to claim 18 wherein said phytase comprises any one or more of the features of claims 1 to 4 and/or claims 5 to 8 and/or claims 9 to 15 and/or claims 16 to 17 and/or claim 19 or 20 .
26 . A phytase variant according to any one of claims 19 to 20 wherein said phytase comprises any one or more of the features of claims claims 1 to 4 and/or claims 5 to 8 and/or claims 9 to 15 and/or claim 16 or 17 and/or claim 18 .
27 . A nucleic acid encoding a phytase any of claims 1 to 26 .
28 . A vector comprising the nucleic acid of claim 27 .
29 . A host cell comprising the nucleic acid of claim 27 or the vector of claim 28 .
30 . A method of producing a phytase variant of any of claims 1 to 26 in a host cell, comprising
a) transforming a host cell with a DNA construct comprising the nucleic acid encoding the phytase of any of claims 1 to 26 , and
b) cultivating the transformed host cell in a suitable culture medium.
31 . The method according to claim 19 , wherein the host cell is a fungal cell, a bacterial cell or a plant cell.
32 . A phytase prepared by the method according to claim 30 or claim 31 .
33 . An enzyme composition comprising at least one phytase of any of claims 1 to 26 or claim 32 including additionally one or more of a glucoamylase, an alpha-amylase, a protease, a pullulanase, an isoamylase, a cellulase, a hemicellulase, a xylanase, a cyclodextrin glycotransferase, a lipase, a laccase, an oxidase, an esterase, a cutinase, another phytase or any combinations thereof.
34 . The enzyme composition of claim 33 , wherein an alpha-amylase is included.
35 . Use of a phytase according to any of claims 1 to 26 or claim 32 or the enzyme composition of claim 33 or claim 34 in starch liquefaction, saccharification, fermentation or simultaneous saccharification-fermentation.
36 . The use of claim 35 , wherein the starch liquefaction, saccharification, fermentation or simultaneous saccharification-fermentation is for production of fermentation alcohol.
37 . The use of claim 36 , wherein the alcohol is ethanol or butanol.
38 . The use of any one of claims 35 to 37 wherein a substrate is subjected to the starch liquefaction, saccharification, fermentation or simultaneous saccharification-fermentation.
39 . The use of claim 38 , wherein the substrate comprises starch.
40 . The use of claim 38 or claim 39 wherein the substrate comprises a grain or cereal.
41 . The use of claim 40 wherein the grain or cereal is wheat, barley, rye, oats, maize, sorghum, corn gluten meal, Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice, oat hulls, palm kernel, citrus pulp or combinations thereof.
42 . The use of claim 41 wherein the rice comprises rice bran or rice hulls.
43 . Use of at least one phytase according to any of claims 1 to 26 or claim 32 as an additive to a food or feed.
44 . Use of at least one phytase according to any of claims 1 to 26 or claim 32 as an additive to a food or feed containing Distillers Dried Grain Solubles (DDGS).
45 . A method for production of food or animal feed comprising a step of admixing at least one phytase according to any of claims 1 to 26 or claim 32 or an enzyme composition according to claim 33 or claim 34 with another food or feed ingredient to form said food or animal feed.
46 . A method for production of food or animal feed comprising a step of spraying at least one phytase according to any of claims 1 to 26 or claim 32 or an enzyme composition according to claim 33 or claim 34 in liquid form onto said food or animal feed.
47 . A method for production of food or animal feed comprising a step of mixing at least one phytase according to any of claims 1 to 26 or claim 32 or an enzyme composition according to claim 33 or claim 34 as a dry product with said food or animal feed.
48 . A method for production of animal feed comprising a step of admixing at least one phytase according to any of claims 1 to 26 or claim 32 or an enzyme composition according to claim 33 or claim 34 with another food or feed ingredient to form said animal feed.
49 . A method for production of animal feed comprising a step of spraying at least one phytase according to any of claims 1 to 26 or claim 32 or an enzyme composition according to claim 33 or claim 34 in liquid form onto said animal feed.
50 . A method for production of animal feed comprising a step of mixing at least one phytase according to any of claims 1 to 26 or claim 32 or an enzyme composition according to claim 33 or claim 34 as a dry product with said animal feed.
51 . A food or animal feed composition comprising either i) at least one phytase according to any of claims 1 to 26 or claim 32 or an enzyme composition according to claim 33 or claim 34 and/or ii) a food or animal feed produced by the method according to any one of claims 45 to 50 .
52 . An animal feed composition comprising either i) at least one phytase according to any of claims 1 to 26 or claim 32 or an enzyme composition according to claim 33 or claim 34 and/or ii) a food or animal feed produced by the method according to any one of claims 45 to 50 .
53 . Use of at least one phytase according to any of claims 1 to 26 or claim 32 or an enzyme composition according to claim 33 or claim 34 in food or animal feed.
54 . Use of at least one phytase according to any of claims 1 to 26 or claim 32 or an enzyme composition according to claim 33 or claim 34 in an animal feed.
55 . A method of reducing the levels of phosphorus in animal manure, characterized in that an animal is fed with at least one phytase according to any of claims 1 to 26 or claim 32 or an enzyme composition according to claim 33 or claim 34 or a feed prepared by the method according to any one of claims 45 to 50 or a composition according to any one of claims 51 - 52 , and wherein said phytase is in an amount effective in converting phytate contained in said animal feed.
56 . Use of at least one phytase according to any of claims 1 to 26 or claim 32 or an enzyme composition according to claim 33 or claim 34 or a feed prepared by the method according to any one of claims 45 to 50 or a composition according to any one of claims 51 - 52 , in the manufacture of an animal feed to reduce the levels of phosphorus in manure from the animal fed with said phytase polypeptide.
57 . A phytase variant substantially as described herein and with reference to the Figures and/or sequence listings.Join the waitlist — get patent alerts
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