Themostable Phytase Variants

Abstract

The present invention relates to a method for producing phytase variants which has at least 74% identity to a phytase derived from Citrobacter braakii and comprises at least two additional disulfide bonds as compared to this phytase. These phytase variants have modified, preferably improved, properties, such as thermostability, temperature profile, pH profile, specific activity, performance in animal feed, reduced protease sensitiliby, and/or an modified glycosylation pattern. The invention also relates to the variants produced, DNA encoding these phytases, methods of their production, as well as the use thereof, e.g. in animal feed and animal feed additives.

1 - 36 . (canceled) 
     
     
         37 . A method of producing a variant phytase having at least 74% identity to SEQ ID NO:2 and comprising the establishment of at least two disulfide bridges as compared to SEQ ID NO:2, wherein said two disulfide bridges are not among the four naturally occurring ones in positions 77/108, 133/407, 178/187, and 381/390 with the numbering as provided in SEQ ID NO:2. 
     
     
         38 . The method of  claim 37 , wherein the at least two disulfide bridges are established in positions selected from the group consisting of the position pairs: A) 52/99; B) 31/177; C) 46/91; D) 141/199; E) 31/176; F) 59/100; and G) 162/247. 
     
     
         39 . The method of  claim 37 , wherein the first disulfide bridge is established in the position pair A between the residues in positions 52 and 99. 
     
     
         40 . The method of  claim 37 , wherein the second disulfide bridge is established in the position pair B between the residues in positions 31 and 177. 
     
     
         41 . The method of  claim 37 , wherein the number of established disulfide bridges is 2, 3, 4, 5, 6, and/or 7. 
     
     
         42 . The method of  claim 37 , wherein the number of established disulfide bridges is two using the following combinations of position pairs:
 A+B, A+C, A+D, A+E, A+F, A+G, B+C, B+D, B+E, B+F, B+G, C+D, C+E, C+F, C+G, D+E, D+F, D+G, E+F, E+G, and F+G, wherein A means 52/99; B means 31/177; C means 46/91; D means 141/199; E means 31/176, F means 59/100; and G means 162/247.   
     
     
         43 . The method of  claim 37 , wherein the number of established disulfide bridges is three using the following combinations of position pairs:
 A+B+C, A+B+D, A+B+E, A+B+F, A+B+G, A+C+D, A+C+E, A+C+F, A+C+G, A+D+E, A+D+F, A+D+G, A+E+F, A+E+G, A+F+G, B+C+D, B+C+E, B+C+F, B+C+G, B+D+E, B+D+F, B+D+G, B+E+F, B+E+G, B+F+G, C+D+E, C+D+F, C+D+G, C+E+F, C+E+G, C+F+G, D+E+F, D+E+G, D+F+G, and E+F+G, wherein A means 52/99; B means 31/177; C means 46/91; D means 141/199; E means 31/176, F means 59/100; and G means 162/247.   
     
     
         44 . The method of  claim 37 , wherein the number of established disulfide bridges is four using the following combinations of position pairs:
 A+B+C+D, A+B+C+E, A+B+C+F, A+B+C+G, A+B+D+E, A+B+D+F, A+B+D+G, A+B+E+F, A+B+E+G, A+B+F+G, A+C+D+E, A+C+D+F, A+C+D+G, A+C+E+F, A+C+E+G, A+C+E+H, A+C+F+G, A+D+E+F, A+D+E+G, A+D+F+G, A+E+F+G, B+C+D+E, B+C+D+F, B+C+D+G, B+C+E+F, B+C+E+G, B+C+F+G, B+D+E+F, B+D+E+G, B+D+F+G, B+E+F+G, C+D+E+F, C+D+E+G, C+D+F+G, C+E+F+G, C+E+F+H, and D+E+F+G, wherein A means 52/99; B means 31/177; C means 46/91; D means 141/199; E means 31/176, F means 59/100; and G means 162/247.   
     
     
         45 . The method of  claim 37 , wherein the number of established disulfide bridges is five using the following combinations of position pairs:
 A+B+C+D+E, A+B+C+D+F, A+B+C+D+G, A+B+C+E+F, A+B+C+E+G, A+B+C+F+G, A+B+D+E+F, A+B+D+E+G, A+B+D+F+G, A+B+E+F+G, A+B+F+G+H, A+C+D+E+F, A+C+D+E+G, A+C+D+F+G, A+C+E+F+G, A+D+E+F+G, B+C+D+E+F, B+C+D+E+G, B+C+D+F+G, B+C+E+F+G, B+D+E+F+G, and C+D+E+F+G, wherein A means 52/99; B means 31/177; C means 46/91; D means 141/199; E means 31/176, F means 59/100; and G means 162/247.   
     
     
         46 . The method of  claim 37 , wherein the number of established disulfide bridges is six using the combinations:
 A+B+C+D+E+F, A+B+C+D+E+G, A+B+C+D+F+G, A+B+C+E+F+G, A+B+D+E+F+G, A+C+D+E+F+G, and B+C+D+E+F+G, wherein A means 52/99; B means 31/177; C means 46/91; D means 141/199; E means 31/176, F means 59/100; and G means 162/247.   
     
     
         47 . The method of  claim 37 , wherein the number of established disulfide bridges is seven using the following combination of position pairs:
 A+B+C+D+E+F+G, wherein A means 52/99; B means 31/177; C means 46/91; D means 141/199; E means 31/176, F means 59/100; and G means 162/247.   
     
     
         48 . The method of  claim 37 , wherein the amino acids in position 179, 180, 181, 182, 183, 184, 185, and 186 have been replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV, or KTDKL. 
     
     
         49 . The method of  claim 37 , comprising a combination of modifications selected from the following: 1*/2*, 1*/2*/3*, 55D/331K, 111 P/241 Q, 179K/180E/181K/182H/183Q/184*/185*/186*, 179K/180E/181K/182Q/183Q/184*/185*/186*, 179K/180E/181K/182K/183V/184*/185*/186*, 179K/180T/181D/182K/183L/184*/185*/186*, 114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L, and 114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L. 
     
     
         50 . The method of  claim 37 , wherein the substitutions to establish the disulfide bridges are:
 A′. G52C/A99C,   B′. N31C/T177C,   C′. W46C/Q91C,   D′. K141C/V199C,   E′. N31C/E176C,   F′. G59C/F100C, and/or   G′. S162C/S247C.   
     
     
         51 . The method of  claim 37 , wherein the mature part of the phytase of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:9 is used as a parent/backbone for producing a phytase variant. 
     
     
         52 . A phytase variant produced by the method of  claim 37 . 
     
     
         53 . A composition comprising the phytase variant of  claim 52 , and
 (a) at least one fat soluble vitamin;   (b) at least one water soluble vitamin; and/or   (c) at least one trace mineral.   
     
     
         54 . A process for reducing phytate levels in animal manure comprising feeding an animal with an effective amount of the composition of  claim 53 . 
     
     
         55 . A method for the treatment of vegetable proteins, comprising the step of adding the phytase of  claim 52  to at least one vegetable protein or protein source. 
     
     
         56 . A method for producing a fermentation product, comprising (a) fermenting using a fermenting microorganism a carbohydrate containing material in the presence of the phytase variant of  claim 52  and (b) producing the fermentation product or fermentation coproduct from the fermented carbohydrate containing material.