Lipolytic enzymes

Details Lipolytic enzymes Lipolytic enzymes

2001-06-26

2003-01-14

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C426S549000, C510S108000, C510S320000, C536S023200

Reexamination Certificate

active

06506588

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to lipolytic enzymes, methods of using and producing lipolytic enzymes, as well as a nucleic acid sequence encoding lipolytic enzymes.
BACKGROUND OF THE INVENTION
Lipolytic enzymes (such as lipases and phospholipases) are capable of hydrolyzing carboxylic ester bonds in a substrate to release carboxylic acids. They are known to be useful, e.g., in baking and detergents.
A lipase/phospholipase from
Fusarium oxysporum
and its sequence are known. WO 98/26057.
SUMMARY OF THE INVENTION
The inventors have isolated a lipolytic enzyme from
Fusarium sulphureum
. The inventors also isolated the gene encoding the novel lipolytic enzyme and cloned it into an
E. coli
strain.
Accordingly, the invention provides a lipolytic enzyme which may be a polypeptide having an amino acid sequence as the mature peptide shown in SEQ ID NO: 1.
Further, the lipolytic enzyme of the invention may be a polypeptide encoded by the lipolytic enzyme encoding part of the DNA sequence cloned into a plasmid present in
Escherichia coli
deposit number DSM 13539.
The lipolytic enzyme may also be an analogue of the polypeptide defined above which:
i) has at least 85% 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 lipolytic enzyme of the invention may be a polypeptide which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with a complementary strand of the nucleic acid sequence of SEQ ID NO: 1 encoding the mature polypeptide or a subsequence thereof having at least 100 nucleotides.
The nucleic acid sequence of the invention may comprise a nucleic acid sequence which encodes the lipolytic enzyme described above, or it may encode a lipolytic enzyme and comprise:
a) the DNA sequence encoding a mature lipolytic enzyme cloned into a plasmid present in
Escherichia coli
DSM 13539,
b) the DNA sequence encoding a mature lipolytic enzyme shown in SEQ ID NO: 1, or
c) an analogue of the DNA sequence defined in a) or b) which
i) has at least 80% 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 sequence of the invention has 82% homology with the lipase/phospholipase from
Fusarium oxysporum
described above, and the corresponding DNA sequence of the invention shows 77% homology with that of the
F. oxysporum
enzyme.
DETAILED DESCRIPTION OF THE INVENTION
Genomic DNA Source
A lipolytic enzyme of the invention may be derived from a strain of Fusarium, particularly
F. sulphureum
, using probes designed on the basis of the DNA sequences in this specification.
A strain of
Escherichia coli
containing a gene encoding lipolytic enzyme was deposited by the inventors under the terms of the Budapest Treaty with the DSMZ—Deutshe Sammmlung von Microorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig DE, Germany. The deposit date was Jun. 15, 2000, and the accession number was DSM 13539.
Properties of Lipolytic Enzyme
The lipolytic enzyme is able to hydrolyze carboxylic ester bonds and is classified as EC 3.1.1 according to Enzyme Nomenclature 1992, Academic Press, Inc. The enzyme has lipase (triacylglycerol lipase) activity (EC 3.1.1.3) and may also have phospholipase activity.
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 lipolytic enzyme of the invention may be produced by transforming a suitable host cell with a DNA sequence encoding the lipolytic enzyme, 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, e.g. a strain of Aspergillus, Fusarium, Trichoderma or Saccharomyces, particularly
A. niger, A. oryzae, F. graminearum, F. sambucinum, F. cerealis
or
S. cerevisiae
. The production of the lipolytic enzyme 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×Denhardt's solution (Sambrook et al. 1989), 0.5% SDS and 100 &mgr;g/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a random-primed (Feinberg, A. P. and Vogelstein, B. (1983)
Anal. Biochem.
132:6-13),
32
P-dCTP-labeled (specific activity >1×10
9
cpm/&mgr;g) probe for 12 hours at approx. 45° C. The filter is then washed two times for 30 minutes in 2×SSC, 0.5% SDS at a temperature of at least 55° C., more preferably at least 60° C., more preferably at least 65° C., even more preferably at least 70° C., especially at least 75° C.
Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using a x-ray film.
Alignment and Homology
The present invention also includes lipolytic enzymes and nucleotide sequences encoding same that have homology to the disclosed sequences. More preferably, the lipolytic enzymes and the nucleotide sequences of the invention may have homologies to the disclosed sequences of at least 85%, at least 90% or at least 95%, e.g. at least 96%, at least 97%, at least 98%.
For purposes of the present invention, alignments of sequences and calculation of homology scores were done using a Needleman-Wunsch alignment (i.e. global alignment), useful for both protein and DNA alignments. The default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively. The penalty for the first residue in a gap is −12 for proteins and −16 for DNA, while the penalty for additional residues in a gap is −2 for proteins and −4 for DNA. Alignment is from the FASTA package version v20u6 (W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid and Sensitive Sequence Comparison with FASTP and FASTA”, Methods in Enzymology, 183:63-98).
Lipase Activity (LU)
A substrate for lipase is prepared an emulsion of 5% by volume of tributyrin (glycerin tributyrate) using 0.1% gum Arabic as emulsifier. The hydrolysis of tributyrin at 30° C. at pH 7 is followed in a pH-stat titration experiment. One unit of lipase activity (1 LU) equals the amount of enzyme capable of releasing 1 &mgr;mol butyric acid/min at the standard conditions. 1 KLU=1000 LU.
Use of Lipolytic Enzyme
The lipolytic enzyme of the invention can be used in various industrial application of lipolytic enzymes, e.g. in baking, detergents, diglyceride synthesis (EP 307154), acidolysis, interesterification (WO 8802775), ester hydrolysis, oil degumming (JP-A 2-153997, U.S. Pat.

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