Lipolytic enzymes

Details Lipolytic enzymes Lipolytic enzymes

2001-06-26

2003-01-28

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S254210, C435S254300, C435S254600, C435S254700, C435S320100, C536S023200, C536S023700, C536S023740

Reexamination Certificate

active

06511837

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a nucleic acid sequence encoding lipolytic enzymes from
Fusarium culmorum,
as well as a recombinant method of producing the lipolytic enzymes.
BACKGROUND OF THE INVENTION
Lipolytic enzymes (such as lipases and phospholipases) are known to be useful, e.g., in baking and detergents. A lipolytic enzyme from
Fusarium culmorum
CBS 513.94 and its N-terminal sequence are disclosed in U.S. Pat. No. 5,830,736. A lipase/phospholipase from
Fusarium oxysporum
and its sequence are disclosed in WO 98/26057.
The enzyme yield of the wild-type strain is very low, and recombinant productions promises to be an economical way of producing the enzyme.
SUMMARY OF THE INVENTION
The inventors have isolated a gene encoding a lipolytic enzyme from
Fusarium culmorum
CBS 513.94 and cloned it into an
E. coli
strain. Accordingly, the invention provides an isolated DNA sequence encoding a lipolytic enzyme.
The nucleic acid sequence of the invention may comprise a nucleic acid sequence which encodes a lipolytic enzyme and comprises:
a) the DNA sequence encoding a mature lipolytic enzyme cloned into a plasmid present in
Escherichia coli
DSM 13537,
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. The invention also provides a recombinant methods of producing the lipolytic enzyme.
A comparison with full-length prior-art sequences shows that the mature amino acid sequence of the lipolytic enzyme from
Fusarium culmorum
has 84% homology with the lipase/phospholipase from
Fusarium oxysporum
described above, and the corresponding DNA sequences show 79% homology.
DETAILED DESCRIPTION OF THE INVENTION
Genomic DNA Source
The DNA sequence of the invention may be derived of
Escherichia coli
DSM 13537 which contains a gene encoding the lipolytic enzyme.
E. coli
DSM 15357 was deposited by the inventors on Jun. 15, 2000 under the terms of the Budapest Treaty with the DSMZ—Deutshe Sammiung von Microorganismen und Zelikulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig DE, Germany.
Lipolytic Enzyme
The lipolytic enzyme encoded by the DNA sequence of the invention 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.
Further properties of the lipolytic enzyme are described in U.S. Pat. No. 5,830,736.
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 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 an 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. No. 5,264,367), production of lysolecithin (JP patent 2794574, JP-B 6-087751) and in the process described in PCT/DK 00/00109.
Use in Baking
The lipolytic enzyme of the invention can be used in the preparation of dough, bread and cakes, e.g. to improve the elasticity of the bread or cake. Thus, the lipolytic enzyme can be used in a process for making bread, comprising adding the lipolytic enzyme to the ingredients of a dough, kneading the dough and baking the dough to make the bread. This can be done in analogy with WO 9404035 and EP 585988.
Use in Detergent
The variant may be used as a detergent additive, e.g. at a concentration (expressed as pure enzyme protein) of 0.001-10 (e.g. 0.01-1) mg per gram of detergent or 0.001-100 (e.g. 0.01-10) mg per liter of wash liquor.
The detergent

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