Lysophospholipase

Details Lysophospholipase Lysophospholipase

2002-12-04

2004-07-06

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S195000, C435S196000, C435S266000, C435S267000

Reexamination Certificate

active

06759225

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to lysophospholipases (LPL), methods of using and producing them, as well as nucleic acid sequences encoding them.
BACKGROUND OF THE INVENTION
Lysophospholipases (EC 3.1.1.5) are enzymes that can hydrolyze 2-lysophospholids to release fatty acid. They are known to be useful, e.g., for improving the filterability of an aqueous solution containing a starch hydrolysate, particularly a wheat starch hydrolysate (EP 219.269).
N. Masuda et al., Eur. J. Biochem., 202, 783-787 (1991) describe an LPL from
Penicillium notatum
as a glycoprotein having a molecular mass of 95 kDa and a published amino acid sequence of 603 amino acid residues. WO 98/31790 and EP 808,903 describe LPL from
Aspergillus foetidus
and
Aspergillus niger
, each having a molecular mass of 36 kDa and an amino acid sequence of 270 amino acids.
JP-A 10-155493 describes a phospholipase A1 from
Aspergillus oryzae
. The mature protein has 269 amino acids.
SUMMARY OF THE INVENTION
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. coil
strains.
Accordingly, the invention provides a lysophospholipase which may be a polypeptide having an amino acid sequence as the mature peptide shown in one of the following or which can be obtained therefrom by substitution, deletion, and/or insertion of one or more amino acids, particularly by deletion of 25-35 amino acids at the C-terminal:
SEQ ID NO: 2 (hereinafter denoted
A. niger
LLPL-1),
SEQ ID NO: 4 (hereinafter denoted
A. niger
LLPL-2),
SEQ ID NO: 6 (hereinafter denoted
A. oryzae
LLPL-1), or
SEQ ID NO: 8 (hereinafter denoted
A. oryzae
LLPL-2).
Further, the lysophospholipase of the invention may be a polypeptide encoded by the lysophospholipase encoding part of the DNA sequence cloned into a plasmid present in
Escherichia coli
deposit number DSM 13003, DSM 13004, DSM 13082 or DSM 13083.
The lysophospholipase may also be an analogue of the polypeptide defined above which:
i) has at least 70% homology with said polypeptide,
ii) is immunologically reactive with an antibody raised against said polypeptide in purified form,
iii) is an allelic variant of said polypeptide,
Finally, the phospholipase of the invention may be a polypeptide which is encoded by a nucleic acid sequence which hybrdizes under high stringency conditions with one of the following sequences or its complementary strand or a subsequence thereof of at least 100 nucleotides:
nucleotides 109-1920 of SEQ ID NO: 1 (encoding
A. niger
LLPL-1),
nucleotides 115-1914 of SEQ ID NO: 3 (encoding
A. niger
LLPL-2),
nucleotides 70-1881 of SEQ ID NO: 5 (encoding
A. oryzae
LLPL-1), or
nucleotides 193-2001 of SEQ ID NO: 7 (encoding
A. oryzae
LLPL-2).
The nucleic acid sequence of the invention may comprise a nucleic acid sequence which encodes any of the lysophospholipases described above, or it may en-code a lysophospholipase and comprise:
a) the lysophospholipase encoding part of the DNA sequence cloned into a plasmid present in
Escherichia coli
DSM 13003, DSM 13004, DSM 13082 or DSM 13083 (encoding
A. niger
LLPL-1
, A. niger
LLPL-2
, A. oryzae
LLPL-1 and
A. oryzae
LLPL-2, respectively),
b) the DNA sequence shown in SEQ ID NO: 1, 3, 5 or 7 (encoding
A. niger
LLPL-1
, A. niger
LLPL-2
, A. oryzae
LLPL-1 and
A. oryzae
LLPL-2, respectively), or
c) an analogue of the DNA sequence defined in a) or b) which
i) has at least 70% homology with said DNA sequence, or
ii) hybridizes at high stringency with said DNA sequence, its complementary strand or a subsequence thereof.
Other aspects of the invention provide a recombinant expression vector comprising the DNA sequence, and a cell transformed with the DNA sequence or the recombinant expression vector.
A comparison with full-length prior-art sequences shows that the mature amino acid sequences of the invention have 60-69% homology with LPL from
Penicillium notatum
(described above), and the corresponding DNA sequences of the invention show 63-68% homology with that of
P. notatum
LPL.
A comparison with published partial sequences shows that an expressed sequence tag (EST) from
Aspergillus nidulans
(GenBank M965865) of 155 amino acid residues can be aligned with the mature
A. oryzae
LLPL-2 of the invention (604 amino acids) with a homology of 79%.
DETAILED DESCRIPTION OF THE INVENTION
Genomic DNA Source
Lysophospholipases of the invention may be derived from strains of Aspergillus, particularly strains of
A. niger
and
A. oryzae
, using probes designed on the basis of the DNA sequences in this specification.
Strains of
Escherichia coli
containing genes encoding lysophospholipase were deposited by the inventors under the terms of the Budapest Treaty with the DSMZ—Deutsche Sammlung von Microorganismen und Zelikulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig DE as follows:
Designation of
Accession
Source organism
lysophospholipase
number
Date deposited
A. niger
LLPL-1
DSM 13003
18 Aug. 1999
A. niger
LLPL-2
DSM 13004
18 Aug. 1999
A. oryzae
LLPL-1
DSM 13082
8 Oct. 1999
A. oryzae
LLPL-2
DSM 13083
8 Oct. 1999
C-terminal Deletion
The lysophospholipase may be derived from the mature peptide shown in SEQ ID NOS: 2, 4, 6 or 8 by deletion at the C-terminal to remove the &ohgr; site residue; while preserving the lysophospholipase activity. The &ohgr; site residue is described in Yoda et al. Biosci. Biotechnol. Biochem. 64, 142-148, 2000, e.g. S577 of SEQ ID NO: 4. Thus, the C-terminal deletion may particularly consist of 25-35 amino acid residues.
A lysophospholipase with a C-terminal deletion may particularly be produced by expression in a strain of
A. oryzae.
Properties of Lysophospholipase
The lysophospholipase of the invention is able to hydrolyze fatty acyl groups in lysophospholipid such as lyso-lecithin (Enzyme Nomendature EC 3.1.1.5). It may also be able to release fatty acids from intact phospholipid (e.g. lecithin).
Recombinant Expression Vector
The expression vector of the invention typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a selectable marker, a transcription terminator, a repressor gene or various activator genes. The vector may be an autonomously replicating vector, or it may be integrated into the host cell genome.
Production by Cultivation of Transformant
The lysophospholipase of the invention may be produced by transforming a suitable host cell with a DNA sequence encoding the phospholipase, cultivating the transformed organism under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
The host organism is preferably a eukaryotic cell, in particular a fungal cell, such as a yeast cell or a filamentous fungal cell, such as a strain of
Aspergillus, Fusarium, Trichoderma
or Saccharomyces, particularly
A. niger, A. oryzae, F. grominearm, F. sambucinum, F. cerealis
or
S. cerevisiae
, e.g. a glucoamylase-producing strain of
A. niger
such as those described in U.S. Pat. No. 3,677,902 or a mutant thereof. The production of the lysophospholipase in such host organisms may be done by the general methods described in EP 238,023 (Novo Nordisk), WO 96/00787 (Novo Nordisk) or EP 244,234 (Alko).
Hybridization
The hybridization is used to indicate that a given DNA sequence is analogous to a nucleotide probe corresponding to a DNA sequence of the invention. The hybridization conditions are described in detail below.
Suitable conditions for determining hybridization between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5×SSC (standard saline citrate) for 10 min, and prehybridization of the filter in a solution of 5×SSC (Sambrook et al. 1989), 5&

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