Polypeptides Having Phospholipase A Activity and Polynucleotides Encoding Same

Patent metadata

Field	Value
Publication number	US-2016289658-A1
Application number	US-201414777617-A
Country	US
Kind code	A1
Filing date	Mar 21, 2014
Priority date	Mar 21, 2013
Publication date	Oct 6, 2016
Grant date	—

Abstract

The present invention relates to isolated polypeptides having phospholipase A activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

First claim

1 . An isolated polypeptide having phospholipase A activity, selected from the group consisting of:
(a) a polypeptide having at least 80% sequence identity to the mature polypeptide of SEQ ID NO: 2 or to the polypeptide of SEQ ID NO: 3; (b) a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence thereof, or (ii) the full-length complement of (i); (c) a polypeptide encoded by a polynucleotide having at least 65% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or to the cDNA sequence thereof; (d) a variant of the mature polypeptide of SEQ ID NO: 2 or of the polypeptide of SEQ ID NO: 3 comprising a substitution, deletion, and/or insertion at one or more positions; and (e) a fragment of the polypeptide of (a), (b), (c), or (d) that has phospholipase A activity.

2 . The polypeptide of claim 1 , wherein the mature polypeptide corresponds to amino acids 28 to 296 of SEQ ID NO: 2 or amino acids 117 to 296 of SEQ ID NO: 2.

3 . The polypeptide of claim 1 having at least 85% sequence identity to the mature polypeptide of SEQ ID NO: 2 or to the polypeptide of SEQ ID NO: 3.

4 . The polypeptide of claim 1 , which is encoded by a polynucleotide having least 85% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or to the cDNA sequence thereof.

5 . The polypeptide of claim 1 , comprising or consisting of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 2, or amino acids 28 to 296 of SEQ ID NO: 2, or amino acids 117 to 296 of SEQ ID NO: 2 or of the polypeptide of SEQ ID NO: 3.

6 . An isolated polynucleotide comprising:
(a) a nucleotide sequence encoding a polypeptide having phospholipase A activity, wherein said polypeptide comprises the mature polypeptide of SEQ ID NO: 2 or amino acids 28 to 296 of SEQ ID NO: 2, or amino acids 117 to 296 of SEQ ID NO: 2 or the polypeptide of SEQ ID NO: 3; (b) a nucleotide sequence encoding a polypeptide having phospholipase A activity, wherein said polypeptide comprises an amino acid sequence which has at least 80% identity with the mature polypeptide of SEQ ID NO: 2 or with the polypeptide of SEQ ID NO: 3; (c) a nucleotide sequence encoding a polypeptide having phospholipase A activity, wherein the nucleotide sequence comprises the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence thereof; (d) a nucleotide sequence which has at least 80% sequence identity to a nucleotide sequence according to (a), (b), or (c); (e) a nucleotide sequence which hybridizes under high stringency conditions with a complementary strand of a nucleotide sequence according to (a), (b), (c), or (d); (f) a subsequence of a nucleotide sequence according to (a), (b), (c), (d), or (e) having at least 100 nucleotides; (g) a sequence which is degenerate as a result of the degeneracy of the genetic code to a sequence as defined in any one of (a), (b), (c), (d), (e), or (f); or (h) a complementary strand of a nucleotide sequence according to (a), (b), (c), (d), (e), (f) or (g).

7 . A nucleic acid construct or expression vector comprising the polynucleotide of claim 6 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

8 . A recombinant host cell comprising the polynucleotide of claim 6 operably linked to one or more control sequences that direct the production of the polypeptide.

9 . A method of producing the polypeptide of claim 1 , comprising:
(a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

10 . A method of producing a polypeptide having phospholipase A activity, comprising:
(a) cultivating the host cell of claim 8 under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

11 . A composition comprising,
a) the polypeptide of claim 1 ; and b) optionally a further enzyme.

12 . The composition of claim 11 , wherein the further enzyme is an enzyme selected from the group consisting of a phospholipase A1, a phospholipase A2, a phospholipase B, a phospholipase C, and a phospholipase C.

13 .- 15 . (canceled)

16 . A method for reducing the content of phosphorus containing components in an edible oil using phospholipase treatment, comprising contacting said oil with an aqueous solution of the polypeptide of claim 1 , until the phosphorus content of the oil is reduced, and then separating the aqueous phase from the treated oil.

17 . The method according to claim 16 , wherein the oil is selected from crude oil, water degummed oil and acid degummed oil.

18 . The method according to claim 16 , wherein the temperature of the oil is between 60 and 75° C.

19 . The method according to claim 16 , wherein the phospholipase treatment is conducted at a pH between 3.0 to 5.5.

Assignees

• Novozymes As

Inventors

• Borch Kim
• Landvik Sara
• Damstrup Marianne Linde
• Brask Jesper

Classifications

• C12N9/18

  Carboxylic ester hydrolases {(3.1.1)} · CPC title

• C12N9/20 Primary

  Triglyceride splitting, e.g. by means of lipase · CPC title

• C12Y301/01

  Carboxylic ester hydrolases (3.1.1) · CPC title

• C11B3/003

  by enzymes or microorganisms, living or dead · CPC title