Xanthan lyase variants and polynucleotides encoding same

Abstract

The present invention relates to xanthan lyase variants and methods for obtaining xanthan lyase variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

1 . A xanthan lyase variant, comprising an alteration at one or more positions in a chelator-induced instability region selected from the group consisting of:
 i) region 1 corresponding to amino acids 154 to 176 of SEQ ID NO: 2,   ii) region 2 corresponding to amino acids 614 to 658 of SEQ ID NO: 2,   iii) region 3 corresponding to amino acids 731 to 803 of SEQ ID NO: 2,   iv) region 4 corresponding to amino acids 807 to 846 of SEQ ID NO: 2,   v) region 5 corresponding to amino acids 872 to 885 of SEQ ID NO: 2, and   vi) region 6 corresponding to amino acids 903 to 1004 of SEQ ID NO: 2,   wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2.   
     
     
         2 . The xanthan lyase variant of  claim 1 , wherein said variant has at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, or at least 99% sequence identity to SEQ ID NO: 2. 
     
     
         3 . The xanthan lyase variant of  claim 1 , wherein said alteration at one or more positions is selected from the group consisting of alterations in positions: 155, 159, 620, 624, 626, 631, 635, 649, 650, 656, 738, 745, 752, 753, 754, 757, 769, 775, 777, 779, 782, 786, 789, 792, 796, 799, 801, 819, 824, 843, 875, 903, 911, 912, 915, 919, 921, 923, 925, 927, 928, 930, 933, 941, 966, 991 and 998, wherein numbering is according to SEQ ID NO: 2. 
     
     
         4 . The xanthan lyase variant of  claim 1 , wherein said alteration at one or more positions is selected from the group consisting of: Y155E, A159P, K620R, A624E, A626Q, T631N, S635E, S635T, T649V, T649K, Q650G, I656V, G738L, K745R, P752R, P752K, G753E, G753Q, G753S, S754E, S754L, S754Q, S754R, S757D, S757P, S757E, A769D, A769T, A769R, L775A, L775F, L775I, L775M, L775Q, L775S, L775Y, D777R, P779V, Y782I, N786K, G789R, K792W, K792Y, K792V, K792A, N796Q, A799H, D801G, K819R, K824R, A843P, K875T, K875E, T903A, T903Q, A911M, A911V, A911S, A912T, A912I, A912Y, T915S, T915V, T915Q, T919D, T919F, T919G, T921R, T921S, T923D, T923H, T925D, T925Q, T925R, T927K, D928W, Y930F, Y930H, Y930L, D933M, G941D, G941E, A966P, N991D and V998K, wherein numbering is according to SEQ ID NO: 2. 
     
     
         5 . The xanthan lyase variant of  claim 1 , further comprising an alteration at one or more positions in at least one region selected from the group consisting of:
 vii) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2,   viii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2,   ix) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2,   x) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2,   xi) region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2,   xii) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and   xiii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2.   
     
     
         6 . The xanthan lyase variant of  claim 5 , wherein said alteration at one or more positions in at least one region selected from the group consisting of regions 7, 8, 9, 10, 11, 12 and 13 is an alteration at one or more positions selected from the group consisting of: 9, 15, 18, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221, 229, 234, 238, 240, 242, 243, 257, 258, 284, 291, 293, 316, 317, 320, 324, 329, 333, 339, 341, 352, 354, 360, 372, 377, 399, 400, 419, 440, 450, 451, 454, 458, 481, 492, 505, 533, 567, 568, 576, 578, 579, 582, 664, 672, 703, 722, 726, 727, 728, 851, 855, 856, 867, 887, 892, 899, 900, 901, 902, 915, 1008 and 1016, wherein numbering is according to SEQ ID NO: 2. 
     
     
         7 . The xanthan lyase variant of  claim 6 , wherein said alteration at one or more positions in at least one region selected from the group consisting of regions 7, 8, 9, 10, 11, 12 and 13 comprises one or more substitutions selected from the group consisting of: K9R, N15T, T18D, L46D, A58L, S66H, Q89Y, K95E, S100D, N106Y, Q109R, Q109D, Q109F, Q109K, Q109A, K183Q, K183R, V188I, A190Q, A203P, K204R, A221P, E229N, E229S, E229V, I234V, I238W, I238L, I238M, I240W, N242S, G243V, Y257W, R258E, R284G, K291R, A293G, A293P, K316R, R317K, K320R, L324Q, K329R, K333R, L339M, I341P, V352I, S354P, K360G, K360R, Q372H, F377Y, N399K, K400R, F419Y, N440K, D450P, K451E, K451R, A454V, D458S, K481R, A492H, A492L, T505I, L533I, K567R, G568A, S578K, S578N, S578R, S579R, S579K, S582K, T664K, N672D, I703L, I722F, P726Q, T727P, M728V, S851F, K855R, E856D, P867S, K887R, N892Y, N892W, N892F, G899S, I900G, D901A, T902F, N1008D and K1016T, wherein numbering is according to SEQ ID NO: 2. 
     
     
         8 . The xanthan lyase variant of  claim 6 , wherein the variant comprises an alteration at one or more positions selected from the group consisting of positions 624, 631, 635, 649, 656, 738, 752, 753, 754, 757, 769, 775, 777, 800, 801, 843, 875, 911 and 915, and an alteration at one or more positions selected from the group consisting of positions 89, 100, 190, 229, 234, 352, 360, 399, 440, 458, 492, 567, 582, 664, 672, 703, 728, 892, 1008 and 1016 of SEQ ID NO: 2. 
     
     
         9 . The xanthan lyase variant of  claim 8 , wherein the variant comprises one or more substitutions selected from the group consisting of Q89Y, S100D, A190Q, E229S, I234V, V352I, K360G, N399K, N440K, D458S, A492H, A492L, K567R, S582K, T664K, N672D, I703L, M728V, N892Y N1008D and K1016T, and one or more substitutions selected from the group consisting of A624E, T631N, S635E, T649K, I656V, G738L, P752K, P752R, G753E, S754E, S754R, S757D, A769D, L775A, D777R, V800P, D801G, A843P, K875T, A911V and T915A. 
     
     
         10 . The xanthan lyase variant of  claim 1 , wherein said variant has an improved stability in a detergent composition compared to a parent xanthan lyase, e.g. a xanthan lyase with SEQ ID NO: 2. 
     
     
         11 . The xanthan lyase variant of  claim 1 , wherein said variant has a half-life improvement factor (HIF) of >1.0, preferably at least 1.2, such as at least 1.5, e.g. at least 2.0, relative to a parent xanthan lyase, e.g. a xanthan lyase with SEQ ID NO: 2,. 
     
     
         12 . A composition comprising at least one xanthan lyase variant of  claim 1 , wherein said composition is a detergent composition comprising one or more detergent components, or a non-detergent composition such as a drilling fluid. 
     
     
         13 . A method for obtaining or producing a xanthan lyase variant, comprising introducing into a parent xanthan lyasean alteration at one or more positions in a chelator-induced instability region selected from the group consisting of:
 i) region 1 corresponding to amino acids 154 to 176 of SEQ ID NO: 2,   ii) region 2 corresponding to amino acids 614 to 658 of SEQ ID NO: 2,   iii) region 3 corresponding to amino acids 731 to 803 of SEQ ID NO: 2,   iv) region 4 corresponding to amino acids 807 to 846 of SEQ ID NO: 2,   v) region 5 corresponding to amino acids 872 to 885 of SEQ ID NO: 2, and   vi) region 6 corresponding to amino acids 903 to 1004 of SEQ ID NO: 2, wherein numbering is according to SEQ ID NO: 2 and wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2, and recovering said variant.   
     
     
         14 . The method of  claim 13 , wherein said alteration at one or more positions is selected from the group consisting of: Y155E, A159P, K620R, A624E, A626Q, T631N, S635E, S635T, T649V, T649K, Q650G, I656V, G738L, K745R, P752R, P752K, G753E, G753Q, G753S, S754E, S754L, S754Q, S754R, S757D, S757P, S757E, A769D, A769T, A769R, L775A, L775F, L775I, L775M, L775Q, L775S, L775Y, D777R, P779V, Y782I, N786K, G789R, K792W, K792Y, K792V, K792A, N796Q, A799H, D801G, K819R, K824R, A843P, K875T, K875E, T903A, T903Q, A911M, A911V, A911S, A912T, A912I, A912Y, T915S, T915V, T915Q, T919D, T919F, T919G, T921R, T921S, T923D, T923H, T925D, T925Q, T925R, T927K, D928W, Y930F, Y930H, Y930L, D933M, G941D, G941E, A966P, N991D and V998K, wherein numbering is according to SEQ ID NO: 2. 
     
     
         15 . The method of  claim 13 , further comprising introducing into a parent xanthan lyase an alteration at one or more positions in at least one region selected from the group consisting of:
 vii) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2,   viii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2,   ix) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2,   x) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2,   xi) region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2,   xii) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and   xiii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2,   wherein numbering is according to SEQ ID NO: 2.   
     
     
         16 . The method of  claim 13 , wherein said alteration at one or more positions provides a variant having a half-life improvement factor (HIF) of >1.0, preferably at least 1.2, such as at least 1.5, e.g. at least 2.0, relative to a parent xanthan lyase, e.g. a xanthan lyase with SEQ ID NO: 2. 
     
     
         17 . A composition comprising a xanthan lyase variant of  claim 1 . 
     
     
         18 . A method of degrading xanthan gum comprising contacting the xanthan gum with a xanthan lyase variant of  claim 1 . 
     
     
         19 . A method of reducing viscosity of a xanthan gum-containing fluid, the method comprising contacting the xanthan gum-containing fluid with a composition comprising a xanthan lyase variant of  claim 1 .