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 . An isolated nucleic acid comprising a sequence as set forth in SEQ ID NO:3 and variants thereof having at least about 50% identity to SEQ ID NO:3 and encoding a polypeptide having α-galactosidase activity.  
     
     
         2 . The isolated nucleic acid of  claim 1 , comprising a sequence as set forth in SEQ ID NO: 3, sequences substantially identical thereto, and sequences complementary thereto.  
     
     
         3 . An isolated nucleic acid that hybridizes to a nucleic acid of  claim 1  under conditions of high stringency.  
     
     
         4 . An isolated nucleic acid that hybridizes to a nucleic acid of  claim 1  under conditions of moderate stringency.  
     
     
         5 . An isolated nucleic acid that hybridizes to a nucleic acid of  claim 1  under conditions of low stringency.  
     
     
         6 . An isolated nucleic acid having at least about 55% homology to the nucleic acid of  claim 1  as determined by analysis with a sequence comparison algorithm.  
     
     
         7 . An isolated nucleic acid having at least about 60% homology to the nucleic acid of  claim 1  as determined by analysis with a sequence comparison algorithm.  
     
     
         8 . An isolated nucleic acid having at least about 65% homology to the nucleic acid of  claim 1  as determined by analysis with a sequence comparison algorithm.  
     
     
         9 . An isolated nucleic acid having at least 70% homology to the nucleic acid of  claim 1  as determined by analysis with a sequence comparison algorithm.  
     
     
         10 . An isolated nucleic acid having at least about 75% homology to the nucleic acid of  claim 1  as determined by analysis with a sequence comparison algorithm.  
     
     
         11 . An isolated nucleic acid having at least 80% homology to the nucleic acid of  claim 1  as determined by analysis with a sequence comparison algorithm.  
     
     
         12 . An isolated nucleic acid having at least about 85% homology to the nucleic acid of  claim 1  as determined by analysis with a sequence comparison algorithm.  
     
     
         13 . An isolated nucleic acid having at least 90% homology to the nucleic acid of  claim 1  as determined by analysis with a sequence comparison algorithm.  
     
     
         14 . An isolated nucleic acid having at least about 95% homology to the nucleic acid of  claim 1  as determined by analysis with a sequence comparison algorithm.  
     
     
         15 . The isolated nucleic acid of  claim 1 ,  2 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 , or  12 , wherein the sequence comparison algorithm is FASTA version 3.0t78 with the default parameters.  
     
     
         16 . An isolated nucleic acid comprising at least 10 consecutive bases of SEQ ID NO: 3, sequences substantially identical thereto, and sequences complementary thereto.  
     
     
         17 . An isolated nucleic acid having at least about 50% homology to the nucleic acid of  claim 10  as determined by analysis with a sequence comparison algorithm or FASTA version 3.0t78 with the default parameters.  
     
     
         18 . An isolated nucleic acid having at least about 55% homology to the nucleic acid of  claim 10  as determined by analysis with a sequence comparison algorithm or FASTA version 3.0t78 with the default parameters.  
     
     
         19 . An isolated nucleic acid having at least about 60% homology to the nucleic acid of  claim 10  as determined by analysis with a sequence comparison algorithm or FASTA version 3.0t78 with the default parameters.  
     
     
         20 . An isolated nucleic acid having at least about 65% homology to the nucleic acid of  claim 10  as determined by analysis with a sequence comparison algorithm or FASTA version 3.0t78 with the default parameters.  
     
     
         21 . An isolated nucleic acid having at least 70% homology to the nucleic acid of  claim 10  as determined by analysis with a sequence comparison algorithm or FASTA version 3.0t78 with the default parameters.  
     
     
         22 . An isolated nucleic acid encoding a polypeptide having a sequence as set forth in SEQ ID NO: 4, and sequences substantially identical thereto.  
     
     
         23 . An isolated nucleic acid encoding a polypeptide comprising at least 10 consecutive amino acids of a polypeptide having a sequence as set forth in of SEQ ID NO: 4, and sequences substantially identical thereto.  
     
     
         24 . A method of producing a polypeptide having a sequence as set forth in SEQ ID NO: 4, and sequences substantially identical thereto comprising introducing a nucleic acid encoding the polypeptide into a host cell under conditions that allow expression of the polypeptide and recovering the polypeptide.  
     
     
         25 . A method of producing a polypeptide comprising at least 10 amino acids of a sequence as set forth in SEQ ID NO: 4, and sequences substantially identical thereto comprising introducing a nucleic acid encoding the polypeptide, operably linked to a promoter, into a host cell under conditions that allow expression of the polypeptide and recovering the polypeptide.  
     
     
         26 . A nucleic acid probe comprising an oligonucleotide from about 10 to 50 nucleotides in length and having an area of at least 10 contiguous nucleotides that is at least 50% complementary to a nucleic acid target region of the nucleic acid sequence set forth in SEQ ID NO:3 and which hybridizes to the nucleic acid target region under moderate to highly stringent conditions to form a detectable target:probe duplex.  
     
     
         27 . The probe of  claim 26 , wherein the oligonucleotide is DNA.  
     
     
         28 . The probe of  claim 26 , which is at least 55% complementary to the nucleic acid target region.  
     
     
         29 . The probe of  claim 26 , which is at least 60% complementary to the nucleic acid target region.  
     
     
         30 . The probe of  claim 26 , which is at least 65% complementary to the nucleic acid target region.  
     
     
         31 . The probe of  claim 26 , which is at least 70% complementary to the nucleic acid target region.  
     
     
         32 . The probe of  claim 26 , which is at least 75% complementary to the nucleic acid target region.  
     
     
         33 . The probe of  claim 26 , wherein the oligonucleotide comprises a sequence which is 80% complementary to the nucleic acid target region.  
     
     
         34 . The probe of  claim 26 , which is at least 85% complementary to the nucleic acid target region.  
     
     
         35 . The probe of  claim 26 , wherein the oligonucleotide comprises a sequence which is 90% complementary to the nucleic acid target region.  
     
     
         36 . The probe of  claim 26 , which is at least 95% complementary to the nucleic acid target region.  
     
     
         37 . The probe of  claim 26 , which is fully complementary to the nucleic acid target region.  
     
     
         38 . The probe of  claim 26 , wherein the oligonucleotide is 15-50 bases in length.  
     
     
         39 . The probe of  claim 26 , wherein the probe further comprises a detectable isotopic label.  
     
     
         40 . The probe of  claim 26 , wherein the probe further comprises a detectable non-isotopic label selected from the group consisting of a fluorescent molecule, a chemiluminescent molecule, an enzyme, a cofactor, an enzyme substrate, and a hapten.  
     
     
         41 . A nucleic acid probe comprising an oligonucleotide from about 15 to 50 nucleotides in length and having an area of at least 15 contiguous nucleotides that is at least 90% complementary to a nucleic acid target region of the nucleic acid sequence set forth in SEQ ID NO:3 and which hybridizes to the nucleic acid target region under moderate to highly stringent conditions to form a detectable target:probe duplex.  
     
     
         42 . A nucleic acid probe comprising an oligonucleotide from about 15 to 50 nucleotides in length and having an area of at least 15 contiguous nucleotides that is at least 95% complementary to a nucleic acid target region of the nucleic acid sequence set forth in SEQ ID NO:3 and which hybridizes to the nucleic acid target region under moderate to highly stringent conditions to form a detectable target:probe duplex.  
     
     
         43 . A nucleic acid probe comprising an oligonucleotide from about 15 to 50 nucleotides in length and having an area of at least 15 contiguous nucleotides that is at least 97% complementary to a nucleic acid target region of the nucleic acid sequence set forth in SEQ ID NO:3 and which hybridizes to the nucleic acid target region under moderate to highly stringent conditions to form a detectable target:probe duplex.  
     
     
         44 . A polynucleotide probe for isolation or identification of α-galactosidase genes having a sequence which is the same as or fully complementary to at least a portion of SEQ ID NO:3.