Enzymes having alpha-galactosidase activity and methods of use thereof

Abstract

The invention relates to α-galactosidase and to polynucleotides encoding the α-galactosidase. In addition methods of designing new α-galactosidases and method of use thereof are also provided. The α-galactosidases have increased activity and stability at increased pH and temperature.

What is claimed is:  
     
         1 . A method of generating a variant comprising: 
 obtaining a nucleic acid comprising a sequence as set forth in SEQ ID NO: 3, sequences substantially identical thereto, sequences complementary thereto, fragments comprising at least 30 consecutive nucleotides thereof, and fragments comprising at least 30 consecutive nucleotides of the sequences complementary to SEQ ID NO: 3; and    modifying one or more nucleotides in said sequence to another nucleotide, deleting one or more nucleotides in said sequence, or adding one or more nucleotides to said sequence.    
     
     
         2 . The method of  claim 1 , wherein the modifications are introduced by a method selected from the group consisting of error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis and any combination thereof.  
     
     
         3 . The method of  claim 1 , wherein the modifications are introduced by error-prone PCR.  
     
     
         4 . The method of  claim 1 , wherein the modifications are introduced by shuffling.  
     
     
         5 . The method of  claim 1 , wherein the modifications are introduced by oligonucleotide-directed mutagenesis.  
     
     
         6 . The method of  claim 1 , wherein the modifications are introduced by assembly PCR.  
     
     
         7 . The method of  claim 1 , wherein the modifications are introduced by sexual PCR mutagenesis.  
     
     
         8 . The method of  claim 1 , wherein the modifications are introduced by in vivo mutagenesis.  
     
     
         9 . The method of  claim 1 , wherein the modifications are introduced by cassette mutagenesis.  
     
     
         10 . The method of  claim 1 , wherein the modifications are introduced by recursive ensemble mutagenesis.  
     
     
         11 . The method of  claim 1 , wherein the modifications are introduced by exponential ensemble mutagenesis.  
     
     
         12 . The method of  claim 1 , wherein the modifications are introduced by site-specific mutagenesis.  
     
     
         13 . The method of  claim 1 , wherein the modifications are introduced by gene reassembly.  
     
     
         14 . The method of  claim 1 , wherein the modifications are introduced by gene site saturated mutagenesis.