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.

1 . An isolated or recombinant 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, wherein the sequence identity is determined by analysis with a sequence comparison algorithm or by a visual inspection.  
     
     
         2 . The isolated or recombinant 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 . The isolated or recombinant nucleic acid of  claim 1 , wherein the sequence identity is at least about 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.  
     
     
         5 . The isolated nucleic acid of  claim 1 , wherein the sequence comparison algorithm is FASTA version 3.0t78 with the default parameters.  
     
     
         6 . An isolated nucleic acid comprising a sequence having at least about 50% sequence identity to at least 10 consecutive bases of a sequence comprising SEQ ID NO: 3, sequences substantially identical thereto, and sequences complementary thereto.  
     
     
         7 . The isolated or recombinant nucleic acid of  claim 6 , wherein the sequence identity is at least about 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.  
     
     
         8 . An isolated or recombinant nucleic acid encoding a polypeptide comprising a sequence having at least about 50% sequence identity to a sequence selected from the group consisting of: 
 (a) a sequence comprising SEQ ID NO: 4, and sequences substantially identical thereto, wherein the sequence identity is determined by analysis with a sequence comparison algorithm or by a visual inspection; and    (b) a sequence 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, wherein the sequence identity is determined by analysis with a sequence comparison algorithm or by a visual inspection.    
     
     
         9 . An isolated or recombinant polypeptide having at least about 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the polypeptide of  claim 8 .  
     
     
         10 . A purified antibody that specifically binds to a polypeptide comprising a sequence selected from the group consisting of: 
 (a) a sequence comprising SEQ ID NO: 4, and sequences substantially identical thereto; and    (b) a sequence comprising at least 10 consecutive amino acids of the polypeptide as set forth in SEQ ID NO: 4, and sequences substantially identical thereto.    
     
     
         11 . The antibody of  claim 10 , wherein the antibodies are polyclonal.  
     
     
         12 . The antibody of  claim 10 , wherein the antibodies are monoclonal.  
     
     
         13 . A method of producing a polypeptide having a sequence selected from the group consisting of: 
 (a) a sequence comprising SEQ ID NO: 4, and sequences substantially identical thereto; and    (b) a sequence comprising at least 10 amino acids of a sequence as set forth in SEQ ID NO: 4; 
 comprising introducing a nucleic acid encoding the polypeptide into a host cell under conditions that allow expression of the polypeptide and recovering the polypeptide.  
   
     
     
         14 . A method of generating a variant comprising: 
 (a) a sequence comprising SEQ ID NO:3, (b) a sequence that hybridizes under stringent conditions to SEQ ID NO:3, wherein the stringent conditions comprise a wash step comprising 30 minutes at room temperature in a solution comprising 150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na 2 EDTA containing 0.5% SDS, followed by a 30 minute wash in fresh solution at T m −10° C., and the sequence encodes a polypeptide having catalase activity and is at least 30 residues in length, (c) a sequences comprising at least 30 consecutive nucleotides of a sequence as set forth in SEQ ID NO:3, (d) a sequence having at least about 50% sequence identity to a sequence as set forth in SEQ ID NO:3, wherein the sequence encodes a polypeptide having catalase activity, and (e) a sequence complementary (a), (b), (c) or (d); 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.    
     
     
         15 . The method of  claim 14 , 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™ (GSSM™) and any combination thereof.  
     
     
         16 . The method of  claim 14 , wherein the modifications are introduced by error-prone PCR.  
     
     
         17 . The method of  claim 14 , wherein the modifications are introduced by shuffling.  
     
     
         18 . The method of  claim 14 , wherein the modifications are introduced by oligonucleotide-directed mutagenesis.  
     
     
         19 . The method of  claim 14 , wherein the modifications are introduced by assembly PCR.  
     
     
         20 . The method of  claim 14 , wherein the modifications are introduced by sexual PCR mutagenesis.  
     
     
         21 . The method of  claim 14 , wherein the modifications are introduced by in vivo mutagenesis.  
     
     
         22 . The method of  claim 14 , wherein the modifications are introduced by cassette mutagenesis.  
     
     
         23 . The method of  claim 14 , wherein the modifications are introduced by recursive ensemble mutagenesis.  
     
     
         24 . The method of  claim 14 , wherein the modifications are introduced by exponential ensemble mutagenesis.  
     
     
         25 . The method of  claim 14 , wherein the modifications are introduced by site-specific mutagenesis.  
     
     
         26 . The method of  claim 14 , wherein the modifications are introduced by gene reassembly.  
     
     
         27 . The method of  claim 14 , wherein the modifications are introduced by Gene Site Saturated Mutagenesis™ (GSSM™).  
     
     
         28 . The isolated or recombinant polypeptide of  claim 8 , wherein the polypeptide is a thermostable enzyme which is stable to heat, is heat resistant and catalyzes the enzymatic hydrolysis of saccharides, and wherein the enzyme is able to renature and regain activity after exposure to temperatures of from about 60 degrees C. to 105 degrees C.  
     
     
         29 . A method of catalyzing the hydrolysis of saccharides comprising contacting a sample containing saccharides with a polypeptide selected from the group consisting of SEQ ID NO: 4 and sequences having at least 50% homology and having α-galactosidase enzyme activity under conditions with facilitate the hydrolysis of the saccharides.  
     
     
         30 . An assay for identifying functional polypeptide fragments or variants encoded by fragments of SEQ ID NO: 3, and sequences substantially identical thereto, which retain the enzymatic function of the polypeptides of SEQ ID NO: 4, and sequences substantially identical thereto, said assay comprising: 
 contacting the polypeptide of SEQ ID NO: 4, and sequences substantially identical thereto, or polypeptide fragment or variant encoded by SEQ ID NO: 3, with a substrate molecule under conditions which allow said polypeptide or fragment or variant to function, and    detecting either a decrease in the level of substrate or an increase in the level of the specific reaction product of the reaction between said polypeptide and substrate, wherein a decrease in the level of substrate or an increase in the level of the reaction product is indicative of a functional polypeptide or fragment or variant.    
     
     
         31 . 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.  
     
     
         32 . The probe of  claim 31 , wherein the oligonucleotide is DNA.  
     
     
         33 . The probe of  claim 31 , which is at least 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or fully complementary to the nucleic acid target region.  
     
     
         34 . The probe of  claim 31 , wherein the oligonucleotide is 15-50 bases in length.  
     
     
         35 . The probe of  claim 31 , wherein the probe further comprises a detectable isotopic label.  
     
     
         36 . The probe of  claim 31 , 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.  
     
     
         37 . 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%, 95% or 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.  
     
     
         38 . 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.  
     
     
         39 . An enzyme preparation comprising a polypeptide of  claim 8  which is liquid.  
     
     
         40 . An enzyme preparation comprising the polypeptide of  claim 8  which is dry.  
     
     
         41 . A method for modifying small molecules, comprising mixing a polypeptide encoded by a polynucleotide of  claim 1  or fragments thereof with a small molecule to produce a modified small molecule.  
     
     
         42 . The method of  claim 41  wherein a library of modified small molecules is tested to determine if a modified small molecule is present within the library which exhibits a desired activity.  
     
     
         43 . The method of  claim 42  wherein a specific biocatalytic reaction which produces the modified small molecule of desired activity is identified by systematically eliminating each of the biocatalytic reactions used to produce a portion of the library, and then testing the small molecules produced in the portion of the library for the presence or absence of the modified small molecule with the desired activity.  
     
     
         44 . The method of  claim 43  wherein the specific biocatalytic reactions which produce the modified small molecule of desired activity is optionally repeated.  
     
     
         45 . The method of  claim 43  wherein 
 (a) the biocatalytic reactions are conducted with a group of biocatalysts that react with distinct structural moieties found within the structure of a small molecule,    (b) each biocatalyst is specific for one structural moiety or a group of related structural moieties; and    (c) each biocatalyst reacts with many different small molecules which contain the distinct structural moiety.    
     
     
         46 . The method of  claim 44  wherein 
 (a) the biocatalytic reactions are conducted with a group of biocatalysts that react with distinct structural moieties found within the structure of a small molecule,    (b) each biocatalyst is specific for one structural moiety or a group of related structural moieties; and    (c) each biocatalyst reacts with many different small molecules which contain the distinct structural moiety.    
     
     
         47 . A method of generating a variant α-galactosidase comprising: 
 obtaining a nucleic acid comprising a sequence as set forth in 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, thereby generating a variant catalase.    
     
     
         48 . The method of  claim 14 , wherein the sequence has at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:3.  
     
     
         49 . The method of  claim 47 , 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™ (GSSM™) and any combination thereof.  
     
     
         50 .