Polypeptides Having Alpha-Galactosidase Activity and Polynucleotides Encoding Same

Abstract

The present invention relates to methods of releasing galactose from legumes using polypeptides having alpha-galactosidase activity. The invention also relates to polypeptides having alpha-galactosidase activity, polynucleotides encoding the polypeptides, nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing the polypeptides. The invention also relates to compositions comprising the polypeptides of the invention and the use of the polypeptides in animal feed.

1 . A method of releasing galactose from plant based material, comprising treating the plant based material with one or more GH36 polypeptides having alpha-galactosidase activity, wherein the GH36 polypeptide having alpha-galactosidase activity is selected from the group consisting of:
 (a) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 2;   (b) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 5;   (c) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 8;   (d) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 12;   (e) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 14;   (f) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 19;   (g) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 19, wherein the variant has alpha-galactosidase activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;   (h) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f) or (g) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;   (i) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f) or (g) and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and   (j) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f) or (g) having alpha-galactosidase activity and having at least 90% of the length of the mature polypeptide.   
     
     
         2 . The method of  claim 1 , wherein the GH36 polypeptide is obtained or obtainable from the taxonomic order Bacillales, preferably the family Bacillaceae or obtained or obtainable from the taxonomic order Eurotiales, preferably the family Aspergillaceae. 
     
     
         3 . The method of  claim 1 , wherein the GH36 polypeptide comprises or consists of amino acids 1 to 728 of SEQ ID NO: 2, amino acids 1 to 734 of SEQ ID NO: 3, amino acids 1 to 731 of SEQ ID NO: 5, amino acids 1 to 737 of SEQ ID NO: 6, amino acids 1 to 732 of SEQ ID NO: 8, amino acids 1 to 738 of SEQ ID NO: 9, amino acids 1 to 719 of SEQ ID NO: 11 or amino acids 1 to 719 of SEQ ID NO: 12, amino acids 1 to 731 of SEQ ID NO: 14, amino acids 1 to 737 of SEQ ID NO: 15, amino acids 1 to 735 of SEQ ID NO: 18, or amino acids 1 to 735 of SEQ ID NO: 19. 
     
     
         4 . The method of  claim 1 , wherein the plant based material is soybean, wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola, rapeseed (oilseed rape) or pea or in a processed form such as soybean meal, full fat soy bean meal, soy protein concentrate (SPC), fermented soybean meal (FSBM) or any combination thereof. 
     
     
         5 . The method of  claim 1 , wherein the GH36 polypeptide releases at least 19 g galactose per kg soybean meal when performed under the reaction conditions 20 mg polypeptide per kg soybean meal in 10% w/v 0.1 M citric acid-phosphate buffer pH 6.5 incubating at 40° C. for 2 hours. 
     
     
         6 . An isolated polypeptide having alpha-galactosidase activity, selected from the group consisting of:
 (a) a polypeptide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 2;   (b) a polypeptide having at least 82%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 5;   (c) a polypeptide having at least 82%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 8;   (d) a polypeptide having at least 84%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 12;   (e) a polypeptide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 14;   (f) a polypeptide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 19;   (g) a polypeptide encoded by a polynucleotide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1;   (h) a polypeptide encoded by a polynucleotide having at least 82%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 4;   (i) a polypeptide encoded by a polynucleotide having at least 82%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 7;   (j) a polypeptide encoded by a polynucleotide having at least 84%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 12;   (k) a polypeptide encoded by a polynucleotide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 14;   (l) a polypeptide encoded by a polynucleotide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 19;   (m) a variant of SEQ ID NO: 2 wherein the variant has alpha-galactosidase activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;   (n) a variant of SEQ ID NO: 5 wherein the variant has alpha-galactosidase activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;   (o) a variant of SEQ ID NO: 8 wherein the variant has alpha-galactosidase activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;   (p) a variant of SEQ ID NO: 12 wherein the variant has alpha-galactosidase activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;   (q) a variant of SEQ ID NO: 14 wherein the variant has alpha-galactosidase activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;   (r) a variant of SEQ ID NO: 19 wherein the variant has alpha-galactosidase activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;   (s) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q) or (r) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;   (t) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q) or (r) and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and   (u) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q) or (r) alpha-galactosidase activity and having at least 90% of the length of the mature polypeptide.   
     
     
         7 . The polypeptide of  claim 6 , wherein the polypeptide comprises or consists of amino acids 1 to 728 of SEQ ID NO: 2, amino acids 1 to 734 of SEQ ID NO: 3, amino acids 1 to 731 of SEQ ID NO: 5, amino acids 1 to 737 of SEQ ID NO: 6, amino acids 1 to 732 of SEQ ID NO: 8, amino acids 1 to 738 of SEQ ID NO: 9, amino acids 1 to 719 of SEQ ID NO: 11 or amino acids 1 to 719 of SEQ ID NO: 12, amino acids 1 to 731 of SEQ ID NO: 14, amino acids 1 to 737 of SEQ ID NO: 15, amino acids 1 to 735 of SEQ ID NO: 18, or amino acids 1 to 735 of SEQ ID NO: 19. 
     
     
         8 . A composition comprising one or more polypeptides of  claim 6 . 
     
     
         9 . The composition of  claim 8 , further comprising one or more components selected from the group consisting of:
 one or more formulating agents;   one or more additional enzymes; and   one or more microbes.   
     
     
         10 . A granule comprising one or more polypeptides of  claim 6 . 
     
     
         11 . An animal feed additive comprising
 (a) one or more polypeptides of  claim 6 ; and   (b) one or more components selected from the group consisting of:
 one or more vitamins; 
 one or more minerals; 
 one or more amino acids; 
 one or more prebiotics; 
 one or more phytogenics; 
 one or more organic acids; and 
 one or more other feed ingredients. 
   
     
     
         12 . A liquid formulation comprising the polypeptide of  claim 6 . 
     
     
         13 . An animal feed comprising plant based material and one or more polypeptides of  claim 6 . 
     
     
         14 . A method of improving one or more performance parameters of an animal comprising administering to one or more animals one or more polypeptides of  claim 6 . 
     
     
         15 . A method for improving the nutritional value of an animal feed, comprising adding to the feed one or more polypeptides of  claim 6 . 
     
     
         16 . A method of preparing an animal feed, comprising mixing one or more polypeptides of  claim 6 . 
     
     
         17 . (canceled) 
     
     
         18 . A polynucleotide encoding the polypeptide of  claim 6 . 
     
     
         19 . A nucleic acid construct or expression vector comprising the polynucleotide of  claim 18  operably linked to one or more control sequences that direct the production of the polypeptide in an expression host. 
     
     
         20 . A recombinant host cell comprising the polynucleotide of  claim 18  operably linked to one or more control sequences that direct the production of the polypeptide. 
     
     
         21 . A method of producing a polypeptide having alpha-galactosidase activity, comprising:
 (a) cultivating the recombinant host cell of  claim 20  under conditions conducive for production of the polypeptide; and   (b) recovering the polypeptide.