Polypeptides having phospholipase C activity and polynucleotides encoding same
The present invention relates to a method of reducing the phospholipid content in an oil or fat composition and polypeptides having PI-specific phospholipase C activity as well as polypeptides having PC, PE-specific phospholipase C activity and combinations thereof capable of catalyzing this reduction. The invention also relates to polynucleotides encoding the polypeptides, nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.
1. A method for reducing the content of phospholipids in an oil composition, the method comprising
a) contacting an oil composition containing a quantity of phospholipids with a phosphatidylinositol phospholipase C and a phosphatidylcholine and phosphatidylethanolamine-specific phospholipase C polypeptide (“a PC and PE-specific phospholipase C”) under conditions sufficient to hydrolyze the phospholipids to diglyceride and phosphate ester, wherein
i) the phosphatidylinositol phospholipase C has at least 90% sequence identity to amino acid residues 26-322 of SEQ ID NO: 2 or amino acid residues 1-298 of SEQ ID NO: 3, and
(ii) the PC and PE-specific phospholipase C has at least 90% sequence identity to amino acid residues 25-280 of SEQ ID NO: 38 or amino acid residues 36-280 of SEQ ID NO: 38; and
b) separating the phosphate ester from the oil composition.
2. The method of claim 1 , wherein the oil is an edible oil.
3. The method of claim 1 , wherein the oil is selected from the group consisting of crude oil, water degummed oil, caustic refined oil and acid degummed oil.
4. The method of claim 1 , wherein the oil comprises phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidyl inositol (PI).
5. The method of claim 4 , wherein the oil comprises at least 50 ppm phosphorus originating from phosphatidyl inositol (PI).
6. The method of claim 1 , wherein
(a) the phosphatidylinositol phospholipase C has at least 95% sequence identity to amino acid residues 26-322 of SEQ ID NO: 2 or amino acid residues 1-298 of SEQ ID NO: 3; and
(b) the PC and PE-specific phospholipase C polypeptide has at least 95% sequence identity to amino acid residues 25-280 of SEQ ID NO: 38 or amino acid residues 36-280 of SEQ ID NO: 38.
7. The method of claim 1 , wherein
(a) the phosphatidylinositol phospholipase C comprises amino acid residues 26-322 of SEQ ID NO: 2 or amino acid residues 1-298 of SEQ ID NO: 3; and
(b) the PC and PE-specific phospholipase C polypeptide comprises to amino acid residues 25-280 of SEQ ID NO: 38 or amino acid residues 36-280 of SEQ ID NO: 38.
8. The method of claim 1 , wherein
(a) the phosphatidylinositol phospholipase C is a fragment of amino acid residues 26-322 of SEQ ID NO: 2 or a fragment of amino acid residues 1-298 of SEQ ID NO: 3; and
(b) the PC and PE-specific phospholipase C polypeptide is a fragment of amino acid residues 25-280 of SEQ ID NO: 38 or a fragment of amino acid residues 36-280 of SEQ ID NO: 38.
9. The method of claim 1 , wherein the oil is contacted with 0.5-200 mg enzyme protein (EP)/Kg oil of the phosphatidylinositol phospholipase C and with 0.5-200 mg enzyme protein (EP)/Kg oil of the PC and PE-specific phospholipase C.
10. The method of claim 1 , wherein the oil is contacted with 1-25 mg enzyme protein (EP)/Kg oil of the phosphatidylinositol phospholipase C and with 1-25 mg enzyme protein (EP)/Kg oil of the PC and PE-specific phospholipase C.
11. The method of claim 1 , wherein the oil is contacted with 2-15 mg enzyme protein (EP)/Kg oil of the phosphatidylinositol phospholipase C and with 2-15 mg enzyme protein (EP)/Kg oil of the PC and PE-specific phospholipase C.