α-galactosidases and methods for making and using them
View Patent ↗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. A method of catalyzing the hydrolysis of saccoharides comprising contacting a sample containing saccharides with a host cell comprising a polynucleotide encoding (a) a polypeptide having an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO:4, or (b) enzymatically active fragments of (a), wherein the polypeptide or the enzymatically active fragments have α-galactosidase enzyme activity, under conditions which facilitate the hydrolysis of the saccharides; and expressing the polypeptide, thereby catalyzing the hydrolysis of saccharides.
2. A method of catalyzing the hydrolysis of saccharides comprising contacting a sample comprising saccharides with a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4, or active fragments thereof, wherein the polypeptide or the active fragments have α-galactosidase enzyme activity, under conditions which facilitate the hydrolysis of the saccharides, thereby catalyzing the hydrolysis of saccharides.
3. The method of claim 1 , wherein the nucleic acid encoding the polypeptide is mRNA.
4. The method of claim 2 , wherein the polypeptide is an isolated or a recombinant polypeptide.
5. The method of claim 4 , wherein the recombinant polypeptide is generated by expressing a nucleic acid comprising a sequence as set forth in SEQ ID NO:3.
6. The method of claim 5 , wherein the nucleic acid is a recombinant nucleic acid and is contained in an expression vector.
7. The method of claim 6 , wherein the expression vector is in a host cell.
8. The method of claim 7 , wherein expresion of the polypeptide is by inducing the host cell.
9. The method of claim 1 , wherein the polypeptide has an amino acid sequence having at least 99% sequence identity to the sequence of SEQ ID:4.
10. A method of catalyzing the hydrolysis of a saccharide comprising the following steps:
(a) providing a sample comprising a saccharide;
(b) providing a polypeptide having an α-galactosidase enzyme activity, wherein the polypeptide comprises (i) a sequence having at least 95% sequence identity to SEQ ID NO: 4, or (ii) enzymatically active fragments of (i); and,
(c) contacting the sample with the polypeptide under conditions which facilitate α-galactosidase enzyme activity, thereby catalyzing the hydrolysis of the saccharide.
11. A method of catalyzing the hydrolysis of a saccharide comprising the following steps:
(a) providing a sample comprising a saccharide;
(b) providing a vector comprising a nucleic acid sequence encoding a polypeptide having an α-galactosidase enzyme activity, wherein the polypeptide comprises (i) a sequence having at least 95% sequence identity to SEQ ID NO: 4, or (ii) enzymatically active fragments of (i);
(c) expressing the polypeptide; and,
(d) contacting the sample with the polypeptide under conditions which facilitate α-galactosidase enzyme activity, thereby catalyzing the hydrolysis of the saccharide.
12. The method of claim 10 , wherein the polypeptide is produced in a cell-free translation system.
13. The method of claim 10 , wherein the polypeptide is produced in a cell-based system.
14. The method of claim 11 , wherein the polypeptide has an amino acid sequence as set forth in SEQ ID NO:4.
15. The method of claim 10 or claim 11 , wherein the saccharide is selected from the group consisting of a polysaccharide and an oligosaccharide.
16. The method of claim 15 , wherein the polysaccharide or oligosaccharide is contained in a legume.
17. The method of claim 15 , wherein the polysaccharide or oligosaccharide is selected from the group consisting of a raffinose, a stachyose and a verbascose.
18. The method of claim 10 or claim 11 , wherein the polypeptide has an amino acid sequence having at least 99% sequence identity to the sequence of SEQ ID NO:4.
19. A method of catalyzing the hydrolysis of a saccharide comprising the following steps:
(a) providing a sample comprising a saccharide;
(b) providing a cell-free translation system comprising a vector comprising a nucleic acid sequence encoding a polypeptide having an α-galactosidase enzyme activity, wherein the polypeptide comprises (i) a sequence having at least 95% sequence identity to SEQ ID NO: 4, or (ii) enzymatically active fragments of (i);
(c) expressing the polypeptide; and,
(d) contacting the sample with the polypeptide under conditions which facilitate α-galactosidase enzyme activity, thereby catalyzing the hydrolysis of the saccharide.
20. The method of claim 19 , wherein the polypeptide has an amino acid sequence having at least 99% sequence identity to the sequence of SEQ ID NO:4.