AU2002311005B2 - Lipoxygenase - Google Patents

Description

WO 02/086114 PCT/DK02/00251

LIPOXYGENASE

FIELD OF THE INVENTION The present invention relates to a lipoxygenase and a polynucleotide encoding it.

BACKGROUND OF THE INVENTION Lipoxygenase (EC 1.13.11.12) is an enzyme that catalyzes the oxygenation of polyunsaturated fatty acids such as linoleic acid, linolenic acid and arachidonic acid, which contain a cis,cis-1,4-pentadiene unit and produces hydroperoxides of these fatty acids. The enzyme is widely distributed in plants and animals. A number of lipoxygenase genes have been isolated from various plant and mammalian sources.

On the other hand, only a limited number of microbial lipoxygenases are known, and no lipoxygenase gene of microbial origin has been described. Su and Oliw, J. Biological Chemistry, 273 13072-79 (1998) describe a lipoxygenase from Gaeumannomyces graminis.

SUMMARY OF THE INVENTION The inventors have found a novel fungal lipoxygenase and determined its sequence, which can be used for the production of the enzyme in industrial scale. They have cloned the gene into E. coli and deposited the clone.

Accordingly, the invention provides a lipoxygenase which is: a) a polypeptide encoded by a DNA sequence cloned into plasmid pUC19 present in Escherichia coli deposited as DSM 14139, b) a polypeptide having an amino acid sequence as the mature peptide shown in SEQ ID NO: 1, or which can be obtained therefrom by substitution, deletion, and/or insertion of one or more amino acids, c) an analogue of the polypeptide defined in or which: i) has at least 50 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, or d) a polypeptide encoded by DNA that hybridizes under low stringency conditions with a complementary strand of i) the DNA sequence cloned into plasmid pUC19 present in Escherichia coli deposited as DSM 14139 or ii) the DNA sequence of SEQ ID NO: 1 encoding the mature polypeptide or a subsequence thereof having at least 100 nucleotides.

The invention also provides a polynucleotide which comprises: WO 02/086114 PCT/DK02/00251 a) the partial DNA sequence encoding a mature lipoxygenase cloned into a plasmid present in Escherichia coli DSM 14139, b) the partial DNA sequence encoding a mature lipoxygenase shown in SEQ ID NO: 1, c) an analogue of the sequence defined in a) or b) which encodes a lipoxygenase and i) has at least 60 homology with said DNA sequence, or ii) hybridizes at high stringency with a complementary strand of said DNA sequence or a subsequence thereof having at least 100 nucleotides, iii) is an allelic variant thereof, or d) a complementary strand of b) or c).

Other aspects of the invention provide a nucleic acid construct and a recombinant expression vector comprising the polynucleotide, a recombinant host cell comprising the construct or the vector, and a method of producing a lipoxygenase by cultivating the cell. Further, the invention provides a method of screening a eukaryotic library to obtain a lipoxygenase and an oligonucleotides probe useful for screening. Finally, the invention provides use of the lipoxygenase in baking and in a detergent.

DETAILED DESCRIPTION OF THE INVENTION Genomic DNA source A lipoxygenase gene of the invention may be derived from a filamentous fungus, e.g.

an Ascomycota, particularly Magnaporthaceae, such as a strain of Magnaporthe, particularly Magnaporthe salvinii Cattaneo (Mycologia 64 110 (1972)). The species is also known under the synonyms Curvularia sigmoidea, Helminthosporium sigmoideum, Leptosphaeria salvinii, Nakataea sigmoidea, Sclerotium oryzae and Vakrabeeja sigmoidea. An example is the strain M. salvinii IFO 6642.

Alternatively, the gene may be isolated from Pyricularia, e.g. P. oryzae or P. grisea, e.g. P. oryzae IFO 30517. The IFO strains are available on commercial terms from Institute for Fermentation, Osaka (IFO), 17-85, Juso-honmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan.

The lipoxygenase gene may be isolated from these organisms using probes designed on the basis of the DNA sequences in this specification.

A strain of Escherichia coli containing a lipoxygenase gene from M. salvinii IFO 6642 was deposited by the inventors under the terms of the Budapest Treaty with the DSMZ Deutsche Sammmlung von Microorganismen und Zellkulturen GmbH, Mascheroder Weg Ib, D- 38124 Braunschweig DE, Germany. The deposit date was 28 February 2001, and the accession number was DSM 14139.

WO 02/086114 PCT/DK02/00251 Production of lipoxygenase by cultivation of transformant The lipoxygenase of the invention may be produced by transforming a suitable host cell with a DNA sequence encoding the lipoxygenase, cultivating the transformed organism under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.

The host organism may be 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 lipoxygenase 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).

Properties of LOX The lipoxygenase of the invention is able to oxidize a wide range of substrates containing a cis-cis-pentadienyl moiety. Thus, it acts on polyunsaturated fatty acids such as linoleic acid (18 carbon atoms, 2 double bonds), linolenic acid arachidonic acid (20:4), eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6). It also acts on substrates other than fatty acids, such as methyl linoleate and probably also triglycerides. The enzyme has a very low Michaelis constant (KM) for linoleic acid and a high specificity (Vmax/KM) towards this substrate.

The lipoxygenase from M. salvinii is a 9-lipoxygenase, i.e. it oxidizes the double bond between carbon atoms 9 and 10 in linoleic acid and linolenic acid.

The lipoxygenase from M. salvinii has optimum activity around pH 7, and it is highly active over a broad pH range 3-12, having more than 50 of optimum activity in the range pH 6-11. It is stable after overnight incubation at pH 5-11.

The native lipoxygenase from M. salvinii has optimum activity at 50-60 0 C. It is quite active at 40-60 0 C, and the activity begins to decline at 70 OC. The lipoxygenase is stable after minutes incubation at pH 7 at temperatures up to 50 0

C.

The reaction rate for recombinant lipoxygenase (expressed in A. oryzae) increases nearly ten times at the optimal temperature for catalysis compared to the rate obtained at room temperature. The maximum reaction rate is obtained at 67.5°C. A steep decrease in rate constant is seen above the temperature optimum. It is believed that glycosylation renders the recombinant enzyme more stable towards heat than the wild-type enzyme.

The recombinant lipoxygenase is quite stable at temperatures up to 50"C for at least one hour. The activity drops in a linear fashion at higher temperatures between 50-600C, and no activity is detected after incubations above 60°C for one hour. No activity loss is detected during incubation at temperatures below WO 02/086114 PCT/DK02/00251 Frozen solutions of the lipoxygenase lose some activity during storage. With addition of 10 glycerol there is no discernible activity loss after two weeks storage at -20 0 C, and the enzyme survived repeated cycles of thaw-freeze without loss of activity.

The lipoxygenase of the invention has good stability in the presence of anionic surfactants. Thus, the lipoxygenase from M. salvinii is stable in the presence of 400 ppm of LAS (linear alkyl-benzene sulfonate).

Use of lipoxygenase The lipoxygenase can be used for green flavor synthesis, e.g. nonenal from 9hydoperoxide of linolenic acid. The synthesis may be done in analogy with Whitehead et al.1995, Cereal foods world 40(4), 193-197 and US 4769243.

The lipoxygenase can also be used for plant hormone synthesis as described in JP H11-29410.

Also the lipoxygenase is a good oxidant of carotenoids, so it can be used for bleaching of foodstuffs such as flour, oil or marine food including carotenoids or carotenoid-like pigments.

The oxidation activity can be utilized for cross-linking of protein, oil, starch, fiber and mixture of these. Cross-linking of chemical compounds can be utilized for synthesis of polymer to give plastic fiber or plastic resin. It can be used for bleaching as a detergent for phenolic, carotenoid or fatty stains or dinginess. Or it can be used for bleaching of waste water or textile dye.

Lipoxygenase can be used for bleaching of plant or marine food materials containing of carotenoids. Thus it could be used for bleaching of flour for bread, noodle or pasta, or bleaching of fish meat or fish oil containing astaxanthin.

It also can be used for cross-linking of protein, oil, starch, plant-fiber or mixture of these in presence of fatty acid, oil or fats. It is useful to change the texture or physical properties of foodstuff or to control of flavor for fat and oil, or to produce polymers made of natural staff beside food use. Cross-linked compounds can be chemical compounds, e.g. phenolic, carbonyl, carboxyl or amide compounds or mixture of these. It could be used for synthesis of plastic fiber or resin.

Other usages of lipoxygenase can be the synthesis of flavor compound such as hexanal or hexenal together as synergy effect of hydroperoxide lyase. Or in case plant material is used as the source of above two enzymes, lipoxygenase can be added to it to improve the yield of flavor compound. The similar can be done for synthesis of plant or animal hormones.

Finally it can be used as bleaching agent. It can be used in detergents for bleaching of phenolic, carotenoid, fatty stains or dinginess of clothes. Or it can be used for bleaching of textile dye or dye for pulp industry in waste water or changing of dye texture.

4 WO 02/086114 PCT/DK02/00251 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 lipoxygenase of the invention may be produced by transforming a suitable host cell with a DNA sequence encoding the lipoxygenase, cultivating the transformed organism under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.

The host organism may be 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 lipoxygenase 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).

The enzyme can be purified in one step by cation-exchange chromatography to homogeneity.

Nucleotide probe A nucleotide probe may be designed on the basis of the DNA sequence of SEQ ID NO: 1 or the polypeptide sequence of SEQ ID NO: 2, particularly the mature peptide part. The probe may be used in screening for LOX-encoding DNA as described below.

A synthetic oligonucleotide primer may be prepared by standard techniques as described in Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual 2 nd edn.) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) on the basis of the mature part of the amino acid sequence in SEQ ID NO: 2 or the corresponding part of the DNA sequence. It may be a degenerate probe and will typically contain at least 20 nucleotides.

Screening of eukaryotic DNA library A polypeptide with lipoxygenase activity may be obtained by a method comprising: a) preparing a eukaryotic DNA library, b) screening the library to select DNA molecules which hybridize to the probe described above, WO 02/086114 PCT/DK02/00251 c) transforming host cells with the selected DNA molecules, d) cultivating the transformed host cells to express polypeptides encoded by the DNA molecules, and e) assaying the expressed polypeptides to select polypeptides having lipoxygenase activity.

The eukaryotic DNA library may be prepared by conventional methods. It may include genomic DNA or double-stranded cDNA derived from suitable sources such as those described above.

Molecular screening for DNA sequences may be carried out by polymerase chain reaction (PCR) followed by hybridization.

In accordance with well-known procedures, the PCR fragment generated in the molecular screening may be isolated and subcloned into a suitable vector. The PCR fragment may be used for screening DNA libraries by e.g. colony or plaque hybridization.

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 may be done at low, medium or high stringency. One example of hybridization conditions is 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 in 5 x SSC (standard saline citrate) for 10 min, and prehybridization of the filter in a solution of 5 x SSC (Sambrook et al. 1989), 5 x Denhardt's solution (Sambrook et al.

1989), 0.5 SDS and 100 pg/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), 3P-dCTP-labeled (specific activity 1 x 109 cpm/pg probe for 12 hours at approx. 450C. The filter is then washed two times for minutes in 2 x SSC, 0.5 SDS at a temperature of at least 55°C, particularly at least more particularly at least 65°C, e.g. at least 700C, or at least Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using an x-ray film.

Alignment and homology The lipoxygenase and the nucleotide sequence of the invention may have homologies to the disclosed sequences of at least 75 or at least 85 particularly at least 90 or at least 95 e.g. at least 98 For purposes of the present invention, alignments of sequences and calculation of 6 WO 02/086114 PCT/DK02/00251 homology scores were done using a Needleman-Wunsch alignment 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 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).

EXAMPLES

Materials and Methods Molecular cloning techniques are described in Sambrook et al. (1989).

The following commercial plasmids and E. coli strains were used for sub-cloning and DNA library construction: pT7Blue (Novagen) pUC19 (TOYOBO, Japan) E. coli JM109 (TOYOBO, Japan) E. coli DH12S (GIBCO BRL, Life Technologies, USA) Labeling and detection of hybridization probe was done using DIG-labeling and detection Kit (Boehringer Manheim). Nylon membrane Hybond-N+ (Amersham, England) was used for DNA transfer for colony hybridization.

Soybean lipoxygenase (type I-B) (cat.# L7315) and astaxanthin (cat.# A-9335) was supplied by Sigma. b-carotene (cat.# 031-05533) were supplied by Wako.

Media and buffer solution COVE-ar: per liter 342.3 g sucrose, 20 ml COVE salt solution, 10 mM acryl amide, mM CsCI 2 30 g Agar noble (Difco) COVE2-ar: per liter 30 g sucrose, 20 ml COVE salt solution, 10 mM acrylamide, 30 g Agar noble (Difco) COVE salt solution: per liter 26 g KCI, 26 g MgSO 4 -7HzO, 76 g KHzPO 4 50ml Cove trace metals.

Cove trace metals: per liter 0.04 g NaB 4 0 7 -10H 2 0, 0.4 g CuSO 4 -5H 2 0, 1.2 g FeSO 4 7H 2 0, 0.7 g MnSO 4

-H

2 0, 0.7 g Na 2 Mo0 2 -2H 2 0, 0.7 g ZnSO 4 -7H 2 0.

AMG trace metals: per liter 14.3 g ZnSO 4 -7H 2 0, 2.5 g CuSO 4 -5H 2 0, 0.5 g NiCI 2 13.8 g FeSO 4 8.5 g MnSO 4 3.0 g citric acid.

YPG: per liter 4 g yeast extract, 1 g KH 2

PO

4 0.5 g MgSO 4 -7H 2 0, 15 g glucose, pH WO 02/086114 PCT/DK02/00251 STC: 0.8 M Sorbitol, 25 mM Tris pH 8, 25 mM CaC 2 STPC: 40% PEG4000 in STC buffer.

Cove top agarose: per liter 342.3 g sucrose, 20 ml COVE salt solution, 10 mM Acetamide, 10 g low melt agarose.

MS-9: per liter 30 g soybean powder, 20 g glycerol, pH MDU-2Bp: per liter 45 g maltose-IH 2 0, 7 g yeast extract, 12 g KHzPO 4 1 g MgSO 4 7H 2 0, 2 g K 2 S0 4 5 g Urea, 1 g NaCI, 0.5 ml AMG trace metal solution pH Host organism Aspergillus oryzae BECh2 is described in WO 00/39322. It is a mutant of JaL228 (described in W098/123000), which is a mutant of IFO4177.

Transformation of A. orvzae Aspergillus oryzae strain BECh2 was inoculated in 100 ml of YPG medium and incubated at 32 0 C for 16 hours with stirring at 80 rpm. Grown mycelia was collected by filtration followed by washing with 0.6 M KCI and re-suspended in 30 ml of 0.6 M KCI containing Glucanex® (Novozymes) at the concentration of 30 pl/ml. The mixture was incubated at 320C with the agitation at 60 rpm until protoplasts were formed. After filtration to remove the remained mycelia, protoplasts were collected by centrifugation and washed with STC buffer twice. The protoplasts were counted with a hematitometer and re-suspended in a solution of STC:STPC:DMSO to a final concentration of 1.2 x 107 protoplasts/ml. About 4 pg of DNA was added to 100 pl of protoplast solution, mixed gently and incubated on ice for minutes. 1 pl STPC buffer was added to the mixture and incubated at 37 0 C for another minutes. After the addition of 10 ml of Cove top agarose pre-warmed at 500C, the reaction mixture was poured onto COVE-ar agar plates. The plates were incubated at 320C for 5 days.

SDS-PAGE

SDS polyacrylamide electrophoresis was carried out using the commercialized gel PAGEL AE6000 NPU-7.5L with the apparatus AE-6400 (Atto, Japan) following the provided protocol. 15 pl of sample was suspended in 15 pl of 2x conc. of sample loading buffer (100 mM Tris-HCI (pH 200 mM Dithiothreitol, 4% SDS, 0.2% Bromophenol blue and 20% glycerol) and boiled for 5 minutes. 20 pl of sample solution was applied to a polyacrylamide gel, and subjected for electrophoresis in the running buffer (25 mM Tris, 0.1% SDS, 192 mM Glycine) at 20 mA per gel. Resulting gel was stained with SYPRO Orange and detected by molecular Imager FX (BIO-RAD).

WO 02/086114 PCT/DK02/00251 Assays for lipoxygenase activity Spectrophotometric assay Lipoxygenase activity was determined spectrophotometrically at 250C by following the formation of hydroperoxides with the absorbance at 234 nm. To 0.98 ml of the buffer mM KH 2 PO4/NaHPO 4 pH 10pl of substrate solution (10mM linolenic acid dispersed with 0.2% Tween20) was added and the reaction was started by the addition of 10 pl of enzyme solution. One unit causes an increase in absorbance at 234 nm of 0.001/min.

FOX assay The assay was initiated by the addition of 20 pl enzyme solution to 80 l of 50 mM each buffer containing 0.7 mM linolenic acid dispersed with 0.02% of Tween 20 using Hiscotron, and incubated for 10 min. The assay was terminated by the addition of 900 pl of FOX reagent: sulfuric acid (25 mM), xylenol orange(100 pM), iron(ll) sulfate (100 pM), butylated hydroxytoluen (4 mM) in methanol:water Blanks contained only substrate solution during the incubation, but enzyme solution was added after the addition of FOX reagent. The yellow color of acidified xylenol orange was converted to a blue color by the lipid hydroperoxidemediated oxidation of Fe2+ ions with the dye. Absorbance of the Fe 3 complex at 620 nm was measured 1 hour after the addition of FOX reagent.

Bleaching assay Bleaching effect by lipoxygenase was examined spectrophotometrically at 25°C by following the absorbance at 470 nm. The pigment solution was prepared as follows. 150 ul of stock pigment solution (1mg each pigment in ml chloroform) was evaporated to be dry. Then ml of the buffer (50 mM KH 2 PO4/NaHPO 4 pH 7.0) with 0.3% of Tween 20 was added slowly and the pigment was dissolved. To 0.98 ml of the pigment solution, 10pl of substrate solution (10mM linolenic acid dispersed with 0.2% of Tween20) was added and the reaction was started by the addition of 10 pl of enzyme solution.

Example 1: Cloning of genomic LOX gene from M. salvinii Genomic DNA from Magnaporthe salvinii was digested with Sac I and separated on agarose gel. Around 2.5 kbp of DNA digestion was recovered from the gel and ligated with BAP treated pUC19 linearized by Sac I. Ligation mixture was transformed into E. coil DH12S to construct a partial genomic library. It was screened, and a lipoxygenase-positive E.

coli colony was isolated and the plasmid, termed pSG28, was recovered. The plasmid pSG28 contained a 2.5 kbp Sacl genomic fragment that contained the presumed LOX homologue sequence. The sequence of 1973 bp out of 2.5 kbp is shown as SEQ. ID 1.

Introns were identified and are indicated in SEQ ID NO: 1. The splice sites were pre- 9 WO 02/086114 PCT/DK02/00251 dicted as described in S.M. Hebsgaard et al., Nucleic Acids Research, 1996, Vol. 24, No. 17, 3439-3452.

The presumed open reading frame consisted of 1851 bp, and the deduced amino acid sequence corresponded to 617 amino acids, shown as SEQ ID NO: 2. The molecular mass was estimated as 67500 Da.

The E. coli DH12S harboring plasmid pSG28 was deposited at DSMZ as DSM 14139 with the accession date 2001-02-28.

Example 2: Expression of M. salvinii LOX in A. oryzae Construction of expression plasmid The partial genomic sequence of M. salvinii genomic gene was amplified by PCR using pSG28 as a template. Primer 3 and 4 (SEQ ID NO: 3 and 4) were designed to make BamH I and Xho I sites at both ends of the PCR product (nucleotides 4-9 of primer 3 and 5-10 of primer 4, respectively). PCR reaction mixture comprised of 2.5 mM dNTP, 30 pmol each of primer 3 and 4, 5 units of LA taq polymerase (Takara) and supplied GC buffer I. Reaction condition was shown below. LA taq polymerase was added to the reaction mixture after step 1.

Step Temperature Time 1 98 °C 10 mins 2 96 °C 20 sec 3 55 °C 45 sec 4 72 °C 30 sec 72 °C 10 mins Step 2 to Step 4 were repeated 30 times.

PCR amplified 1.9 kb fragment was isolated and cloned into pT7Blue resulting in pSG29.

The plasmid pSG29 was digested by BamHII and Xhol and 1.9 kb of fragment which contained the LOX gene was ligated with pMT2188 digested with BamHI and Xhol. The plasmid pMT2188 has a modified Aspergillus niger neutral amylase promoter, Aspergillus nidulans TPI leader sequence, Aspergillus niger glucoamylase terminator, Aspergillus nidulans amdS gene as a marker for fungal transformation and S. cerevisiae ura3 as the marker for E. coli transformation. Transformation was done with E. coli DB6507 in which pyrF gene is deficient and can be complemented with S. cerevisiae Ura3. Resulting plasmid was termed WO 02/086114 Expression of M. salvinii LOX in A. oryzae PCT/DK02/00251 A. oryzae BECh2 was transformed with the plasmid pSG30 and selection positive transformants were isolated. Transformants were grown on COVE 2 -ar at 320C for 5 days and inoculated to 100 ml of MS-9 shaking flask. After the cultivation with vigorous agitation at 32°C for 1 day, 3 ml of each culture was transferred to 100 ml of MDU-2Bp in shaking flask to cultivate at 32°C for 3 days. Culture broth was centrifuged at 3500 rpm for 10 minutes and supernatant was collected.

Lipoxygenase activities of the supernatant were determined spectrophotometrically as described before. Positive transformants showed about 100,000U/ml culture broth while untransformed A. oryzae BECh2 showed no activity. Culture supernatant was also subjected to SDS-PAGE analysis. Positive transformants showed 80-100kDa smear band which indicated the protein was heavily glycosylated. Untransformed A. oryzae BECh2 did not show any significant bands.

Example 3: Substrate specificity of lipoxygenase Kinetic parameters for a number of substrates were determined by standard methods for the M. salvinii lipoxygenase.

Vmax KM Vmax/KM Substrate (pmol/min/mg) (pM) (pmollminlmglpM) Linoleic acid 2.63 1 2.557 Na linoleate 2.07 0.41 5.061 Linoelaidic acid No activity No activity No activity Linolenic acid 1.9 0.4 4.488 Eicosadienoic acid 2.02 11 0.177 Arachidonic acid 2.44 5.5 0.446 Linoleoyl chloride 0.97 12 0.080 Methyl linoleate 0.82 30 0.026 Linoleoyl acetate 0.77 9 0.085 Linoleoyl alcohol 1.4 8 0.175 WO 02/086114 PCT/DK02/00251 For comparison, one substrate was also tested with soybean lipoxygenase.

Subse Vmax KM Vmax/KM Substrate (pmol/min/mg) (pM) (pmol/min/mg/pM) Linoleic acid 12.3 230 0.054 Example 3: pH dependence of lipoxygenase activity The relative activity of the M. salvinii lipoxygenase at various pH values was determined by the FOX assay described above, using the following buffers: 50 mM citric acid/sodium citrate (pH 2.21-3.73) KH 2 PO4/Na 2

HPO

4 (pH 5.30,6.17), Tris/HCI (pH 7.01,8.02), glycylglycine NaCI/NaOH (pH 9.33-11.0).

pH Relative Activity 2.21 7.11 2.90 20.6 3.73 27.7 5.30 60.0 6.17 83.7 7.01 100 8.02 92.9 9.33 82.6 11.0 77.7 Example 4: Temperature dependence of lipoxygenase activity The effect of temperature on the M. salvinii lipoxygenase was studied by 10 min incubation at pH Temperature Relative Activity 50.1 90.0 100 99.6 60.4 WO 02/086114 PCT/DK02/00251 Example 5: Bleaching effect of lipoxygenases The bleaching effect of M. salvinli LOX was examined. Soybean LI was included for comparison. 1-carotene and astaxanthin were used as pigments.

1-carotene Time (min) M. salvinil Soybean LI 0 0.3783 0.3575 0.4 0.3791 0.3616 0.8 0.3729 0.3601 1.2 0.3702 0.362 1.4 0.3685 0.3602 1.8 0.3651 0.3602 2.2 0.3633 0.3595 2.6 0.3486 0.3595 3 0.341 0.3594 AA470/min 0.0121 0.00005 LOX activity 2.652 1.962 Astaxanthin Time (min.) M. salvinii Soybean LI 0 0.5292 0.5026 0.4 0.5244 0.5029 0.8 0.5177 0.505 1 0.51 66 0.5025 1.4 0.512 0.5013 1.8 0.5004 0.4993 2.2 0.4876 0.4985 2.6 0.4714 0.4986 WO 02/086114 PCT/DK02/00251 0.4566 0.498 AA47O1min 0,0239 0.0021 LOX activity 2.4952 2.018 The results show that M. salvinfi LOX bleaches the pigment solutions. Soybean LOX showed little effect on bleaching.

WO 02/086114 PCT/DK02/00251 0-1 Form PCTIRO/134 (EASY) Indications Relating to Deposited Microorganism(s) or Other Biological Material (PCT Rule 13bis) 0-1-1 Prepared using PCT-EASY Version 2.92 (updated 01.01.2002) 0-2 International Application No.

0-3 Applicant's or agent's file reference 10148 1 The indications made below relate to the deposited microorganism(s) or other biological material referred to in the description on: 1-1 page 2 1-2 line 36-37 1-3 Identification of Deposit 1-3-1 Name of depositary institution DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH 1-3-2 Address of depositary Institution Mascheroder Weg Ib, D-38124 Braunschweig, Germany 1-3-3 Dateofdeposit 28 February 2001 (28.02.2001) 1-3-4 Accession Number DSMZ 14139 1-4 Additional Indications NONE Designated States for Which all designated States Indications are Made 1-6 Separate Furnishing of Indications NONE These indications will be submitted to the International Bureau later FOR RECEIVING OFFICE USE ONLY

Claims (26)

1. A lipoxygenase which is: a) a polypeptide encoded by a DNA sequence cloned into plasmid pUC19 present in Escherichia coli deposited as DSM 14139, b) a polypeptide having an amino acid sequence as the mature peptide shown in SEQ ID NO: 1, or which can be obtained therefrom by substitution, deletion, and/or insertion of one or more amino acids, c) an analogue of the polypeptide defined in or which: i) has at least 50% of homology with said polypeptide, 0t ii) is immunologically reactive with an antibody raised against said polypeptide in purified form, iii) is an allelic variant of said polypeptide, or d) a polypeptide encoded by DNA that hybridizes under low stringency conditions with a complementary strand of i) the DNA sequence cloned into plasmid pUC19 present in Escherichia coli deposited as DSM 14139 or ii) the DNA sequence of SEQ ID NO: 1 encoding the mature polypeptide or a subsequence thereof having at least 100 nucleotides.

2. The lipoxygenase of claim 1 which is: an analogue of the polypeptide defined in or which: i) shares at least 85% homology with said polypeptide, ii) is immunologically reactive with an antibody raised against said polypeptide in purified form, or iii) is an allelic variant of said polypeptide; or a polypeptide encoded by DNA that hybridizes under high stringency conditions with a complementary strand of i) the DNA sequence cloned into plasmid pUC19 present in Escherichia coli deposited as DSM 14139 or ii) the DNA sequence of SEQ ID NO: 1 encoding the mature polypeptide or a subsequence thereof having at least 100 nucleotides.

3. The lipoxygenase of claim 1 or claim 2 which is: a polypeptide encoded by a DNA sequence cloned into plasmid pUC19 present in Escherichia coli deposited as DSM 14139; or AH21(926509 1) AAK a polypeptide having an amino acid sequence as the mature peptide shown in SEQ ID NO: 1.

4. The lipoxygenase of any one of claims 1 to 3 which is derived from a filamentous fungus.

5. The lipoxygenase of claim 4, which is derived from a member of the Ascomycota.

6. The lipoxygenase of claim 4, which is derived from a species of Magnaporthe

7. The lipoxygenase of claim 6, which is derived from a strain ofM. salvinii.

8. The lipoxygenase of claim 7, which is derived from M. salvinii strain IFO io 6642.

9. DNA comprising a nucleic acid sequence which encodes the lipoxygenase of any one of claims 1 to 8. A polynucleotide which comprises: a) the partial DNA sequence encoding a mature lipoxygenase cloned into a plasmid present in Escherichia coli DSM 14139, b) the partial DNA sequence encoding a mature lipoxygenase shown in SEQ ID NO: 1, c) an analogue of the sequence defined in a) or b) which encodes a lipoxygenase and i) has at least 60% homology with said DNA sequence, or ii) hybridizes at high stringency with a complementary strand of said DNA sequence or a subsequence thereof having at least 100 nucleotides, iii) is an allelic variant thereof, or d) a complementary strand of b) or c).

11. A polynucleotide of claim 10, which comprises an analogue of the sequence defined in a) or b) which encodes a lipoxygenase and i) has at least 85% homology with said DNA sequence, or ii) hybridizes at high stringency with a complementary strand of said DNA sequence or a subsequence thereof having at least 100 nucleotides, or iii) is an allelic variant thereof, or a complementary strand of said analogue.

12. A polynucleotide of claim 10, which comprises the partial DNA sequence encoding a mature lipoxygenase cloned into a plasmid present in Escherichia coli DSM 14139, All21(926509_] AAK (ii) the partial DNA sequence encoding a mature lipoxygenase shown in SEQ ID NO: 1,or (iii) a complementary strand of(i) or (ii).

13. A nucleic acid construct comprising the nucleic acid sequence of any one of claims 10 to 12 operably linked to one or more control sequences capable of directing the expression of the lipoxygenase in a suitable expression host.

14. A nucleic acid construct comprising a nucleic acid sequence encoding a lipoxygenase as defined in claim 1, substantially as hereinbefore described with reference to any one of the examples. 1o 15. A recombinant expression vector comprising the nucleic acid construct of claim 13 or claim 14, a promoter, and transcriptional and translational stop signals.

16. A recombinant expression vector comprising a nucleic acid sequence encoding a lipoxygenase as defined in claim 1, substantially as hereinbefore described with reference to any one of the examples.

17. A recombinant host cell comprising the nucleic acid construct of claim 13 or claim 14 or the vector of claim 15 or claim 16.

18. A recombinant host cell comprising a nucleic acid sequence encoding a lipoxygenase as defined in claim 1, substantially as hereinbefore described with reference to any one of the examples.

19. A method for producing a lipoxygenase comprising cultivating the host cell of claim 17 or claim 18 under conditions conducive to production of the lipoxygenase, and recovering the lipoxygenase. A method for producing a lipoxygenase as defined in claim 1, substantially as hereinbefore described with reference to any one of the examples.

21. An oligonucleotide probe which consists of at least 20 nucleotides and which encodes a partial polypeptide sequence of SEQ ID NO: 2.

22. A method for obtaining a polypeptide with lipoxygenase activity, comprising: a) preparing a eukaryotic DNA library, b) screening the library to select DNA molecules which hybridize to the probe of claim 21, c) transforming host cells with the selected DNA molecules, d) cultivating the transformed host cells to express polypeptides encoded by the DNA molecules, and e) assaying the expressed polypeptides to select polypeptides having lipoxygenase activity. All21(')250') 1) AAK

23. A method for preparing a dough or a baked product made from dough, comprising adding the lipoxygenase of any one of claims 1 to 8 to the dough.

24. A dough composition comprising the lipoxygenase of any one of claims 1 to 8.

25. A detergent composition comprising a surfactant and the lipoxygenase of any one of claims 1 to 8.

26. The detergent composition of claim 25 wherein the surfactant is anionic.

27. A process for oxidizing a polyunsaturated fatty acid comprising contacting the acid with the lipoxygenase of any one of claims 1 to 8 in the presence of air.

28. Use of the process of claim 27 for green flavor synthesis or plant hormone synthesis. Dated 3 September, 2007 Novozymes A/S Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON AH121(92(50)_ I) AAK WO 02/086114 WO 02/86114PCT/DK02/00251

10148-WO. SEQUENCE LISTING <110> NovozymeS A/S <120> Lipoxygenase <130> 10148-wo <160> 4 <170> Patentln version 3.1 <210> 1 <211> 1973 <212> DNA <213> Magnaporthe salvinji <220> <221> CDS <222> .(381) <223> <220> <221> mat-peptide <222> 0 <223> <220> <221> CDS <222> (1970) <223> -<400> 1 atg cgc atc gga ctc ttg gcc ttc gcc gtc gcg gcg cgc tat gtg gaa 48 met Arg Ile G ly Leu LeU Ala Phe Ala Val Ala Ala Arg Tyr Vat Glu -10 Page 1 WO 02/086114 WO 02/86114PCT/DK02/00251 10148-WO. gcg ctg cca gtc gcg agc g gc gaa gaa gtq gcc Ala LeU Pro \ial Ala Ser Gly Glu Glu ValI Ala -1 1 5 10 acg acg ctg ccc tcg acg tcg agc agc tct gcg Thr Thr Leu Pro Ser Thr Ser Ser Ser Ser Ala 25 aag tac acg ctt ccc cac gag gac ccc aac ccg Lys Tyr Thr Leu Pro His Glu Asp Pro Asn Pro 40 gag ata gcg tta aag agg gg a gg g ttc ctc tac Glu Ilie Ala Leu Lys Arg Gly Sly Phe Leu Tyr 55 gg c cag act acc ttt tac ccc agc ggg acc ctg Gly Gin Thr Thr Phe Tyr Pro Ser Gly Thr Leu 70 caa cgc gac cag gcc ctc tgg ctc aqg gat gca Gln Arg Asp Gln Ala Leu Trp Leu Arg Asp Ala 85 90 aca gcg tat cgt gaa gcc aac gag aca ctg agg Thr Ala Tyr Arg Glu Ala Asn Glu Thr Leu Arg 100 105 gtatgtgtcg agccgtgttt atgcgttcca atcattctct ccggggttac agccaagccg attcagtagc taactcggaa tcg tcg tcC gct ccg Ser Ser Sen Ala Pro ctt ccc tcc ccg acc Leu Pro Sen Pro Thr gaa gcg agg aag gcc GlU Ala Arg LYS Ala gg a ccc tcc acc ctg Gly Pro sen Thr Leu gg g acc gcc atg tcg Gly Thr Ala met Ser gag aac caa acg ata GIU Asn Gin Thr Ile gat atc cag agc Asp Ilie Gin Ser 110 gtgctcctgt ccgtccccgc tgtctggttt gctctgcag cat gygc g gt His Gly G] y cat tgg aaa. His Tnp Lys aac tac act Asn Tyr Thr 145 ccc tac agc: Pro Tyr Ser 160 agc gtc gag Ser Val Glu 175 gcg ctc cac Ala Leu His aag aaa tac Lys Lys Tyr ctt ttc tat Leu Phe Tyr 225 ctc aag acg ctt LeU LYS Thr Leu 115 gcg tcg gtc cca Ala ser Val Pro 130 tcg gac ctg ctc Ser Asp Leu Leu ctc aag cgc ctc LeU Lys Arg Leu 165 gac aag gtq gtc ASP LYS ValI Val 180 aag gcc g gc cgt Lys Ala Gly Arg 195 acg ccg cag gca Thr Pro Gln Ala 210 gtg gac gcg cgg Val Asp Ala Arg gac gac ttc gcg ctc ctc tac ASP ASP Phe Ala LeU Leu Tyr 120 gag g ga ata gaa aag ggc atg Glu Gy Ilie GlU Lys Gly met 135 140 ttt tcc atg gag cgg ctc tcc Phe Sen met Glu Arg Leu Ser 150 155 cat cca acc aag gac aag ctg His Pro Thr Lys Asp Lys Leu 170 aag cag ctg acg gcc acg acg LYS Gln LeU Thr Ala Thr Thr 185 ctc ttc ttc gtt gac cac: agc Leu Phe Phe Val ASP His Ser 200 g gt cgg tat gct gcg gcc tgc Gly Ang Tyr Ala Ala Ala Cys 215 220 tcc aat cag ttc ctg ccg ctg Ser Asn Gin Phe Leu Pro Leu 230 235 ctg acg tac acg cca ctc gac: Leu Thr Tyr Thr Pro Leu Asp gac g gc Asp G ly 125 ctg agc Leu Ser aac aac Asn Asfl ccg ttc Pro Phe ctt gcg Leu Ala 190 gat cag ASP Gin 205 cag g gg Gln G ly gcc atc Ala lie gac aag ASP LYS aag acc LYS Thr 240 aac gtyggc gca Asn ValG Y Ala page 2 WO 02/086114 WO 02/86114PCT/DK02/00251 10148-WO. aac Asn 255 tac Tyr cac His gtg gaa Gl U ctg Leu 335 9cg Ang ccc Pro atg met g cg Al a 415 g cg Al a ctg Leu cac His ctc LeU gtc Val 495 tcg Ser ctg Leu tac Tyr 275 atc Ile atc Ile ttc Phe g cg Al a ttc Phe 355 gac ASP gtc Val gag 61 U gac Asp 435 ctg Leu cag Gin g ct Al a gac Asp gt 515 gcc aag Ala Lys 260 cat gtc His Val cgg acg Ang Thr atg tat Met Tyr aac ccg ASfl Pro 325 gcc gtc Ala Val 340 cgg gcc Arg Ala gac acc ASP Thr gcg cag Ala Gln gac tcg Asp Ser 405 ctg cag Leu Gln 420 ttc ccc Phe Pro acg cac Thr His ctt tac Leu Tyr 485 g c ctg 6Y Leu tcg ctg Ser Leu atc atg ttc Ilie met Phe ctg ttc cac Leu Phe His 280 cta agc gag Leu Ser GlU 295 caa gcc tat Gin Ala Tyr 310 gac ttt ctc ASP Phe Leu 360 ttt g gc g gc Phe 6 ly Gly 375 cgc gtc ctc Arg Val Leu 390 acc tac gqgg Thr Tyr Gly gac tgg g t ASP Trp Va acg gcg ccg Thr Ala Pro 440 ata gcc tgg Ile Ala Trp 455 ccc gtg cgc Pro Val Arg 470 gcg ccc gtc Ala Pro V'al ctg gcg tgg Leu Ala Trp ctg gcg cgc LeU Ala Arg S20 aac aac aat gac ASp gaa Gi U gt 300 cca Pro aac Asfl tac Tyr ctg Leu ccg Pro 380 cgc A rg aac Asn cgc Arg 460 aag LYS gag 61 U ttc Ph e 270 gty g cc Al a cat His 350 aag LYS ttc Phe ttc Phe ctg Leu ccg Pro 430 gag GIU cac His ccg Pro gcc Al a aaa Lys 510 980 1028 1076 1124 1172 1220 1268 1316 1364 1412 1460 1508 1556 1604 1652 1700 1748 ttc aac cgc gcg cag gtt Phe Asn Arg Ala Gin Val 525 Page 3 WO 02/086114 WO 02/86114PCT/DK02/00251 agg gac aga aac cag acg gtg Arg ASP Aflg Asn Gin Thr Val 530 ctg gct gga aat g gc gag gcg Leu Ala Gly Asn Gly Giu Ala 545 gag gag acg g gc cgg ata agc Glu Glu Thr Gly Ang Ile Ser 560 565 agc aag g gc ctg agc cag gqg Sen Lys Gy1y Leu Ser Gin G] y 575 580 ccc gcg gtg aac ccg ttt ttc Pro Ala val ASfl Pro Phe Phe 595 <210> 2 <211> 617 <212> PRT <213> magnaporthe saivinij <400> 2 Met Arg Ala LeU I Thr Thr I Lys Tyr- GiU Ilie Gly Gin- Gin Arg Thr Al a cgc A rg tac Ty r 550 cgc Arg atg Met ctg Leu Phe Gi u Sen ASP Gi y Sen Leu Gi u 10148-WO. aac atg ttc gcc gca ccg gag ctg Asn met Phe Al a Ala Pro Giu Leu 535 540 gcg gcg gcc aac gcg agg ttc gtc Ala Ala Ala Asn Ala Ang Phe Val 555 gag ata gag g gc agg g gt ttc gat GlU Ilie Giu Gi y Arg Gi y Phe ASP 570 ccc ttt atc tgg acc gcc ttg aat Pro Phe Ile Trp Thr Ala Leu Asn 585 590 agc atc tag Se Ilie 600 1796 1844 1892 1940 1973 Gly Gly Leu Lys Thr Leu Asp Asp Phe Ala LeU LeU Tyr ASP Gly His 115 120 125 Page 4 WO 02/086114 WO 02/86114PCT/DK02/00251 Trp LYS Tyr Thr 145 Tyr Ser 160 Val GlU Leu His Lys Tyr Phe Tyr 225 Thr Asn 240 ASP Trp Ser Gin Met Ala Leu Asn 305 Arg le 320 Pro Ala Glu Gly Gly Leu Phe Tyr 385 Ala Sen Val Pro GlU 130 Ser ASP LeU LeU Phe 150 LeU Lys Arg LeU His 165 Asp Lys Val Val Lys 180 Lys Ala Gly Arg Leu 195 Thr Pro Gin Ala Gly 210 Val ASP Ala Arg Ser 230 Val Gly Ala Asp Leu 245 Leu Leu Ala Lys Ile 260 Met Tyr His Val Leu 275 Ala Ile Arg Thr Leu 290 Ag Ilie met Tyr Gin 310 Leu Phe Asn Pro Gly 325 Thr Ala Ala Val Asp 340 Gly Phe Arg Ala Gly 355 Val Gly Asp Thr Phe 370 Giu Asp Ala Gin Arg Gi y 135 Ser Pro Gi n Ph e Arg 215 As n Th r Met Ph e Se r 295 Al a Gi y Phe Ty r Gi y 375 10148-WO. Ilie GlU LYS Gly met Giu Arg Leu 155 Thr LYS ASP LYS 170 LeU Thr Ala Thr 185 Phe Val ASP His 200 Tyr Ala Ala Ala Gin Phe Leu Pro 235 Tyr Thr Pro Leu 250 Phe Asn Asn Asn 265 His Thr Val Pro 280 GlU ser His Pro Tyr Ala Ile Afl9 315 Phe Trp ASP Gin 330 Leu Ser Ser le 345 Val GiU Asn Asn 360 Gly Pro Ala Leui Met 140 Ser Leu Th r Se r Cys 220 Leu ASP Asp GIU Val 300 Pro Asn Tyr Leu Pro 380 Sen Asn Phe Al a 190 Gin Gi y 3lie Lys Phe 270 Val Al a Gi y Gi y His 350 LYS Phe Val Leu Gly Aia Arg Gly Phe Met Page WO 02/086114 WO 02/86114PCT/DK02/00251 10148-WO. Gin Ala 400 Arg Asp Gin Val Val Gly Val Leu 465 HiS Pro 480 Ala Ser Val Glu Asp Arg Ala Gly 545 GlU Thr 560 Lys Gly Al a Val <210> <211> <212> <213> <220> <221> Thr Tyr Gly Gly Asp 410 Glu Arg Th r se r Al a 490 Pro Asfl Al a Asn Gi y 570 Trp 2-9 DNA rti fi cial/unknown mi sc-feature Page 6 WO 02/086114 WO 02/86114PCT/DK02/00251 10148-WO. <c223> Artificial <220> <221> mi sc-feature <223> Primer 3 <400> 3 cgcggatcca tgcgcatcgg actcttggc <210> <211> <212> <213> <220> <221> <22 3> <220> <221> <223> 4 31 DNA Artificial/unknown mi scfeature Artificial ml sc-featu re Primer 4 <400> 4 cccgctcgag ctagatgctc aggaaaaacg g Page 7