Phytase variants

Abstract

The present invention relates to phytase variants derived from a parent phytase which is homologous to a Peniophora lycii phytase and comprises at least one substitution in at least one of a number of positions corresponding to a position in the Peniophora lycii phytase. The variants are of improved properties, in particular of a higher specific activity and/or more thermostable than the parent phytase, e.g., of a Tm of up to 82° C. at pH 5.5, as determined by DSC, and/or of a doubted specific activity. The invention also relates to DNA encoding the phytase variants, methods of their production, as well as the use thereof, e.g., in animal feed and animal feed additives.

1 . A variant of a parent phytase, comprising a substitution in at least one position of at least one region selected from the group of regions consisting of: 20; 26; 28-31; 37-48; 55-69; 73-83; 91-119; 123-126; 135-142; 152-163; 169-199; 204-222; 229-238; 248-266; 284-289; 300-308; 321-335; 343-350; 384-398; 407-412; and 419-430; wherein
 (a) the variant has phytase activity; and   (b) each position corresponds to a position of SEQ ID NO: 2; and   (c) the variant has a percentage of identity to SEQ ID NO: 2 of at least 75%; and   (d) with the proviso that the variant is not a variant of the  Peniophora lycii  CBS 686.96 phytase selected from the group consisting of: G26S; Y28N; D29S; F31Y; E42D,K,A; T4SR; V46I; W59F; S62D; A64K; R65Q,E,G,A; S66T; R67A,I,Q,K; Q68Y,V,I; K74R,A; A99N; 100S; L102V; P103E; F104L; N107T,Q; S109M; H110S,V; Q111E,N; T112A,S; M116F,I,L; A135S; D137Y; Q138D,S,G; D142A; ( )154fH; S155N; G156P; E1570,S; E169S; E170A,P; G171A; ( )171aS; T174P,A; N177S; N178S,T; M179T,V,L; N182A,V; V184G; D185E; ( )185eT; G186S,A; D187R,K,S; ( )187aV,T; E138Q,S,A,V; S189E; V195L; N199A,P; L204N; A207D,H; S216T; D217E; L219Y,T; T220Y, N; L221F; M222L; L230V; ( )235eE,Q; V248R,I; S249Q,A; E251D; Y254A,L,G; D255N; D257K; Y259F; T262H; P264A; G286R,H; Q287K,S; A288P; T321Q,S,G; M322I; V323I; P324A; A327S,F,I,L; F332Y; N333P; E344R; R346P; 348F; V349L,A,S; D350S,V; G388A; V393R; E395K,T; and L396R.   
     
     
         2 . The variant of  claim 1 , which comprises a substitution or a combination of substitutions selected from the following group of substitutions and combinations of substitutions:
 20R+95R+97V+98G;   29N+102I;   29N+118A:   29N+163P+324S;   29N+163P+324S+395G;   29N+234S;   29N+324S;   29S;   29S+45A+69A+99D+110Y+125D+324A+334E+344S+350V;   29S+63N+125D+183K+218T+324A+334E+350V+421S;   29S+95H+110W+183W+324A+350V;   29S+95M+110W+183W+324A+350V;   29S+95N+110R+183W+324A+350V;   29S+95R+110W+183W+324A+350V;   29S+95S+110R+183W+324A+350V;   29S+95S+110 W+183W+324A+350V;   29S+95T+110R+183W+324A+350V;   29S+95T+110Y+125D+183W+286S+324A+350V;   29S+95T+110Y+152A+156C+157Q+160R+183W+324A+350V;   29S+95T+110Y+156R+157Q+183W+324A+350V;   29S+95T+110Y+157K+183W+185H+187R+190S+191R+248G+324A+350V;   29S+95T+110Y+183W+185A+248A+324A+350V;   29S+95T+110Y+183W+185A+248C+324A+350V;   29S+95T+110Y+183W+185A+248E+324A+350V;   29S+95T+110Y+183W+185A+248H+324A+350V;   29S+95T+110Y+183W+185E+248S+324A+350V;   29S+95T+110Y+183W+185H+248G+321A+323A+324A+350V;   29S+95T+110Y+183W+185H+248G+324A+350V;   29S+95T+110Y+183W+185H+187S+190K+191S+210G+222I+248G+324A+S350V;   29S+95T+110Y+183W+185K+248G+324A+349K+350V;   29S+95T+110Y+183W+185K+248S+324A+350V;   29S+95T+110+183W+185K+248T+324A+349A+350L;   29S+95T+110Y+183W+185K+248T+324A+349L+350L;   29S+95T+110Y+185W+185K+248T+324A+349L+350M;   29S+95T+110Y+183W+185K+248T+324A+350V;   29S+95T+110Y+183W+185P+248G+324A+350V;   29S+95T+110Y+183W+185P+248S+324A+350V;   29S+95T+110Y+183W+191 L+324A+350V;   29S+95T+110Y+185W+248D+324A+350V;   29S+95T+110Y+183W+324A;   29S+95T+110Y+183W+324A+350V;   29S+99D+125D+324A+334E+350V;   29S+110Y+183W+185N+324A+350V;   29S+125D+218T+324A+344S+350V;   29S+125D+234K+324A+330R;   29S+125D+234K+324A+330R+395G;   29S+125D+324A;   29S+125D+324A+330R+395G;   29S+125D+324A+350V;   29S+183A+324A+350V;   29S+183G+324A+334E+344S+350V;   29S+183W+324A+350V;   29S+205S;   29S+324A+350V;   29T+324L;   45A+350V+421R+422R+423N+424S+425L+426E+427G+428R+429I+430M;   69A+334E;   99D+398T;   114A+187G+194R+324S;   218T+334G;   323A+324S;   324S;   324S+328T;   324S+345D+347Q;   324S+395G;   330R;   334D+343N+344V;   334E+344A;   345H+347P+350V; and   305V.   
     
     
         3 . A transgenic plant, or plant part, capable of expressing a phytase variant of  claim 1 . 
     
     
         4 . A transgenic, non-human animal, or products, or elements thereof, capable of expressing a phytase variant of  claim 1 . 
     
     
         5 . A composition comprising at least one phytase variant of  claim 1 , and
 (a) at least one fat soluble vitamin;   (b) at least one water soluble vitamin; and/or   (c) at least one trace mineral.   
     
     
         6 . The composition of  claim 5 , further comprising at least one enzyme selected from the following group of enzymes: xylanases, endoglucanases, endo-1,3(4)-beta-glucanases, and proteases. 
     
     
         7 . The composition of  claim 5 , which is an animal feed additive. 
     
     
         8 . An animal feed composition having a crude protein content of 50 to 800 g/kg and comprising the variant of  claim 1 . 
     
     
         9 . A method for improving the nutritional value of an animal feed, comprising adding a variant of  claim 1  to the feed. 
     
     
         10 . A process for reducing phytate levels in animal manure, comprising feeding an animal with an effective amount of an animal feed composition of  claim 8 . 
     
     
         11 . A method for the treatment of vegetable proteins, comprising adding the variant of  claim 1  to at least one vegetable protein or protein source. 
     
     
         12 . An isolated nucleic acid sequence comprising a nucleic acid sequence which encodes the variant of  claim 1 . 
     
     
         13 . A nucleic acid construct comprising the nucleic acid sequence of  claim 12  operably linked to one or more control sequences that direct the production of the polypeptide in a suitable expression host. 
     
     
         14 . A recombinant expression vector comprising the nucleic acid construct of  claim 13 . 
     
     
         15 . A recombinant host cell comprising the nucleic acid construct of  claim 13 . 
     
     
         16 . A method for producing a variant comprising
 (a) cultivating the host cell of  claim 15  to produce a supernatant comprising the variant; and   (b) recovering the variant.   
     
     
         17 . Use of the variant of  claim 1  in animal feed; in the preparation of animal feed; for improving the nutritional value of animal feed; for reducing phytate levels in animal manure; for the treatment of vegetable proteins; or for liberating phosphorous from a phytase substrate.