Lysophospholipase
The inventors have isolated lysophospholipases from Aspergillus ( A. niger and A. oryzae ) having molecular masses of about 68 kDa and amino acid sequences of 600-604 amino acid residues. The novel lysophospholipases have only a limited homology to known amino acid sequences. The inventors also isolated genes encoding the novel enzymes and cloned them into E. coli strains.
1 - 12 . cancel.
13 . An isolated lysophospholipase, comprising:
a) a polypeptide encoded by a lysophospholipase encoding part of the DNA sequence cloned into a plasmid present in Escherichia coli deposit number DSM 13004;
b) a polypeptide having an amino acid sequence of amino acids 1-458 in SEQ ID NO: 4;
c) an analogue of the polypeptide defined in (a) or (b) which has at least 95% sequence homology with said polypeptide; or
d) a polypeptide which is encoded by a nucleic acid sequence which hybridizes with a complementary strand of the nucleic acid sequence shown as nucleotides 115-1914 of SEQ ID NO:3 under hybridization conditions comprising prehybridizing in a solution of 5×SSC, 5× Denhardt's solution, 0.5% SDS and 100 μg/ml of denatured sonicated salmon sperm DNA, followed by hybridization in the same solution for 12 hours at approx. 45° C., followed by washing in 2×SSC, 0.5% SDS for 30 minutes at a temperature of at least 65° C.
14 . The lysophospholipase of claim 12 which is native to a strain of Aspergillus.
15 . The lysophospholipase of claim 12 , which is native to a strain of A. niger.
16 . The lysophospholipase of claim 12 , comprising a deletion of 25-35 amino acids at the C-terminal end.
17 . The lysophospholipase of claim 12 , comprising a polypeptide that is encoded by a nucleic acid sequence which hybridizes with a complementary strand of the nucleic acid sequence shown as nucleotides 115-1914 of SEQ ID NO:3 under hybridization conditions comprising prehybridizing in a solution of 5×SSC, 5× Denhardt's solution, 0.5% SDS and 100 μg/ml of denatured sonicated salmon sperm DNA, followed by hybridization in the same solution for 12 hours at approx. 45° C., followed by washing in 2 x SSC, 0.5% SDS for 30 minutes at a temperature of at least 65° C.
18 . The lysophospholipase of claim 12 , comprising a polypeptide that is encoded by a nucleic acid sequence which hybridizes with a complementary strand of the nucleic acid sequence shown as nucleotides 115-1914 of SEQ ID NO:3 under hybridization conditions comprising prehybridizing in a solution of 5×SSC, 5× Denhardt's solution, 0.5% SDS and 100 μg/ml of denatured sonicated salmon sperm DNA, followed by hybridization in the same solution for 12 hours at approx. 45° C., followed by washing in 2×SSC, 0.5% SDS for 30 minutes at a temperature of at least 70° C.
19 . The lysophospholipase of claim 12 , comprising a polypeptide that is encoded by a nucleic acid sequence which hybridizes with a complementary strand of the nucleic acid sequence shown as nucleotides 115-1914 of SEQ ID NO:3 under hybridization conditions comprising prehybridizing in a solution of 5×SSC, 5× Denhardt's solution, 0.5% SDS and 100 pg/ml of denatured sonicated salmon sperm DNA, followed by hybridization in the same solution for 12 hours at approx. 45° C., followed by washing in 2×SSC, 0.5% SDS for 30 minutes at a temperature of at least 75° C.
20 . The lysophospholipase of claim 12 , comprising a polypeptide which has at least 95% homology to the polypeptide encoded by a lysophospholipase encoding part of the DNA sequence cloned into a plasmid present in Escherichia coli deposit number DSM 13004 or to the polypeptide having an amino acid sequence of amino acids 1-458 in SEQ ID NO: 4.
21 . The lysophospholipase of claim 12 , comprising a polypeptide which has at least 98% homology to the polypeptide encoded by a lysophospholipase encoding part of the DNA sequence cloned into a plasmid present in Escherichia coli deposit number DSM 13004 or to the polypeptide having an amino acid sequence of amino acids 1-458 in SEQ ID NO: 4.
22 . The lysophospholipase of claim 12 , comprising a polypeptide encoded by a lysophospholipase encoding part of the DNA sequence cloned into a plasmid present in Escherichia coli deposit number DSM 13004.
23 . The lysophospholipase of claim 12 , comprising a polypeptide having an amino acid sequence comprising amino acids 1-458 in SEQ ID NO: 4.
24 . A process for hydrolyzing fatty acyl groups in a phospholipid or lysophospholipid, comprising treating the phospholipid or lysophospholipid with the lysophospholipase of claim 12 .
25 . A process for improving the filterability of an aqueous solution or slurry of carbohydrate origin which contains phospholipid, which process comprises treating the solution or slurry with the lysophospholipase of claim 12 .
26 . The process of claim 28 , wherein the solution or slurry contains a starch hydrolysate.
27 . The process of claim 28 , wherein the solution or slurry contains a wheat starch hydrolysate.