Polynucleotides encoding phytase polypeptides

Abstract

The present invention relates to barley isolated polypeptides having phytase activity, recombinant DNA sequences encoding such polypeptides, methods of producing such polypeptides and the use of said polypeptides in transgenic plants. The invention disclose polypeptides having affinity for the substrate phytate (1,2,3,4,5,6 myo-inositol-hexakisphosphate, phytic acid), comprising an amino acid sequences as described by the invention. Furthermore, the invention discloses DNA fragments encoding said polypeptides, and cDNA fragments encoding said polypeptides. The polypeptides of the invention may for example be used as an additive in animal feeds, an additive in food for human consumption or to extract proteins from rice bran. The invention also concerns a transgenic plant or part thereof, wherein said plant or part thereof have been genetically modified to comprise a polypeptide as defined in by the invention.

1 . An isolated polypeptide having affinity for the substrate phytate (1,2,3,4,5,6 myo-inositol-hexakisphosphate, phytic acid), comprising an amino acid sequence as given in SEQ ID NO:1 or an equivalent thereof, having a sequence identity with SEQ ID NO:1 of at least 70% and at least one or more of the amino acid sequences as given in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:12, or an equivalent thereof having a sequence identify with SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:12 of at least 70%.  
     
     
         2 . A polypeptide according to  claim 1 , capable of binding a monoclonal antibody raised against a polypeptide having the sequence of SEQ ID NO:1.  
     
     
         3 . A polypeptide of  claim 1 , comprising an N-terininal amino acid sequence as given in SEQ ID NO:1 or an equivalent thereof, having a sequence identity of with SEQ ID NO:1 of at least 70%.  
     
     
         4 . A polypeptide of  claim 1 , having a denatured molecular weight of between 50-75 kDa, preferably 53-70 kDa, more preferably 55-68 kDa as determined by SDS-PAGE.  
     
     
         5 . A polypeptide of  claim 1 , having a native molecular weight of 35-55 kDa as determined by gelfiltration.  
     
     
         6 . A polypeptide of  claim 1 , having a substrate affinity of 0.05-0.50 mM.  
     
     
         7 . A polypeptide of  claim 1 , having a V max  of 600-1000 U/mg.  
     
     
         8 . A polypeptide of  claim 1 , having an isoelectric point of between 5.0-8.0.  
     
     
         9 . A polypeptide of  claim 1 , having a pH optimum of between 4 and 8, preferably between 5-7.  
     
     
         10 . A polypeptide of  claim 1 , wherein the activity is optimal at a temperature between 30°-65° C., preferably between 35°-60° C., and more preferably between 40°-55° C.  
     
     
         11 . A polypeptide according to  claim 1 , exhibiting 6-phytase activity.  
     
     
         12 . A DNA fragment encoding a polypeptide as defined in  claim 1 .  
     
     
         13 . A cDNA fragment encoding a polypeptide as defined in  claim 1 .  
     
     
         14 . A cDNA fragment encoding a polypeptide according to  claim 13  having an amino acid sequence as given in SEQ ID NO:8, or an equivalent thereof having a sequence identity with SEQ ID NO:8 of at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95%.  
     
     
         15 . A DNA fragment encoding a polypeptide according to  claim 12  having an amino acid sequence as given in SEQ ID NO: 10 or an equivalent thereof having a sequence identity with SEQ ID NO:10 of at least 80%, such as 90%.  
     
     
         16 . A DNA fragment encoding a polypeptide according to  claim 12  having an amino acid sequence as given in SEQ ID NO:11 or an equivalent thereof having a sequence identity with SEQ ID NO:11 of at least 80%, such as 90%.  
     
     
         17 . An expression cassette comprising a DNA fragment as defined in  claim 12 .  
     
     
         18 . A cell which is capable of expressing a polypeptide and which is transformed with an expression cassette as defined in  claim 17 .  
     
     
         19 . A method of producing a polypeptide having affinity for the substrate phytate (1,2,3,4,5,6 myo-inositol-hexakisphosphate, phytic acid) from a grass or cereal comprising the steps of: 
 short time extraction of the polypeptide,    obtaining an extract,    subjecting the extract to purification steps,    purifying the polypeptide from said extract,    obtaining the polypeptide.    
     
     
         20 . A method according to  claim 19  additionally comprising the steps of: 
 exposing the polypeptide to epitope specific antibodies raised against said polypeptide,    assessing the specificity by Western blotting.    
     
     
         21 . A method of producing a recombinant polypeptide having affinity for the substrate phytate (1,2,3,4,5,6 myo-inositol-hexakisphosphate, phytic acid), comprising culturing a cell as defined in  claim 18 , in a suitable culture medium under conditions allowing expression of the polypeptide, and recovering the polypeptide from the culture.  
     
     
         22 . A product obtained by the method as defined in  claim 19 .  
     
     
         23 . A product obtained by the method as defined in  claim 20 .  
     
     
         24 . Use of a polypeptide as defined in  claim 1 .  
     
     
         25 . The use according to  claim 24  as an additive in animal feeds.  
     
     
         26 . The use according to  claim 24  as an additive in food for human consumption.  
     
     
         27 . The use according to  claim 24  as an industrial processing enzyme.  
     
     
         28 . The use according to  claim 24  for the extraction of protein from rice bran.  
     
     
         29 . The use according to  claim 24  in a transgenic plant or part thereof.  
     
     
         30 . A transgenic plant or part thereof, wherein said plant or part thereof have been genetically modified to comprise a polypeptide as defined in  claim 1 .  
     
     
         31 . A transgenic plant according to  claim 30 , being a plant selected from wheat, barley, rye, spelt, oat, rice or maize.