WO2002024881A1

WO2002024881A1 - Phospholipase from zygoascus hellenicus

Info

Publication number: WO2002024881A1
Application number: PCT/DK2001/000600
Authority: WO
Inventors: Tom Anton Busk Nielsen; Michiyo Ishii; Shamkant Anant Patkar; Noriko Tsutsumi
Original assignee: Novozymes A/S
Priority date: 2000-09-25
Filing date: 2001-09-18
Publication date: 2002-03-28
Also published as: AU2001289588A1

Abstract

The inventors discovered and isolated a phospholipase from Zygoascus hellenicus. The inventors also isolated the gene encoding the novel phospholipase and cloned it into an E. coli strain.

Description

PHOSPHOLIPASE FROM ZYGOASCUS HELLENICUS

FIELD OF THE INVENTION

The present invention relates to a phospholipase, methods of using and producing it, as well as a nucleic acid sequence encoding it.

BACKGROUND OF THE INVENTION

Phospholipases are known to be useful, e.g. in baking and oil degumming.

Fu et al., Microbiology 143, 331-340 (1997) describe the cloning and characterization of a gene (LIP1) which encodes a lipase from the pathogenic yeast Candida albicans and state that the lipase has no phospholipase activity. The GenBank accession number for LIP1 is U34807.

LIP4 (EMBL, GenBank AF 191317) is described as a secreted lipase from Candida albicans.

Molecular cloning and expression of phospholipase B genes from Candida albicans has been described. Sugiyama et al., Medical Mycology, 37 (1), 61-67 (1999). Hoover et al., FEMS Microbiology Letters, 167 (2), 163-169 (1998). Leidich et al., J. Biological Chemistry, 273 (40), 26078-26086 (1998). GenBank AL033501.

SUMMARY OF THE INVENTION

The inventors discovered and isolated a phospholipase from Zygoascus hellenicus. The inventors also isolated and sequenced the gene encoding the novel phospholipase and cloned it into an E. coli strain.

Accordingly, the invention provides a phospholipase which may be a polypeptide having an amino acid sequence as the mature peptide shown in SEQ ID NO: 1 or a polypeptide encoded by the phospholipase encoding part of the DNA sequence cloned into a plas- mid present in E. coli DSM 13648. The phospholipase may also be an analogue of the polypeptide defined above which: i) has at least 50 % identity 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 hybridizes at 55°C 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 phospholipase described above, or it may encode a phospholipase and comprise: a) the DNA sequence encoding a mature phospholipase cloned into a plasmid pre- sent in Escherichia coli DSM 13648, b) the DNA sequence encoding a mature phospholipase shown in SEQ ID NO: 1, or c) an analogue of the DNA sequence defined in a) or b) which i) has at least 60 % identity with said DNA sequence, or ii) hybridizes at 55°C with said DNA sequence, its complementary strand or a sub- sequence 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 known sequences shows that the closest prior-art sequence is LIP4 (EMBL, GenBank AF 191317), described as a secreted lipase from Candida albicans. The identity was calculated to be 38 % for the mature proteins and 52 % for DNA sequences encoding the mature proteins, disregarding the STOP codons.

DETAILED DESCRIPTION OF THE INVENTION

Genomic DNA source A phospholipase of the invention may be derived from a strain of Zygoascus, particularly Z. hellenicus, e.g. strain CBS 4075, using probes designed on the basis of the DNA sequences in this specification. The strain is commercially available from Centraalbureau voor Schimmelcultures, Baarn, the Netherlands.

A strain of Escherichia coli containing a gene encoding phospholipase was depos- ited 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 10 August 2000, and the accession number was DSM 13648. The deposit was made by Novo Nordisk A S and was later assigned to Novozymes A/S.

Properties of phospholipase

The phospholipase has phospholipase A activity and is able to hydrolyze lecithin by releasing fatty acid. It has no lipase activity and shows no activity towards tributyrin. The isoelectric point is around 4.2. 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 phospholipase 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 particularly 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. graminearum, F. sambucinum, F. cerealis or S. cerevisiae, e.g. a glucoamylase-producing strain of A. niger such as those described in US 3677902 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 x SSC (standard saline citrate) for 10 min, and prehy- bridization 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 μg/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), ³²P- dCTP-labeled (specific activity > 1 x 10⁹ cpm/μg ) probe for 12 hours at approx. 45°C. The filter is then washed two times for 30 minutes in 2 x SSC, 0.5 % SDS at a temperature of at least 55°C, more particularly at least 60°C, more particularly at least 65°C, even more particularly 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 identity

The phospholipase and the nucleotide sequence of the invention may have identities to the disclosed sequences of at least 60 % or at least 70 %, e.g. 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 identity scores were done using the software Align, 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 pen- alty for additional residues in a gap is -2 for proteins and -4 for DNA. Align 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).

Use of phospholipase

The phospholipase of the invention can be used in various industrial application of phospholipases, e.g. as described below.

Use in baking

The phospholipase of the invention can be used in the preparation of dough and baked products (such as bread and cakes), e.g. to achieve a larger loaf volume of the baked product and/or a better shape retention during baking and/or to improve the elasticity of the baked product. Thus, the phospholipase can be used in a process for making bread, comprising adding the phospholipase to the ingredients of a dough, kneading the dough and baking the dough to make the bread. This can be done in analogy with US 4567056 or WO 99/53769.

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 composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations. In a laundry detergent, the variant may be effective for the removal of fatty stains, for whiteness maintenance and for dingy cleanup. A laundry detergent composition may be formulated as described in GB 2247025, WO 9901531 or WO 9903962. The detergent composition of the invention may particularly be formulated for hand or machine dishwashing operations, e.g. as described in GB 2,247,025 (Unilever) or WO 99/01531 (Procter & Gamble). In a dishwashing composition, the variant may be effective for removal of greasy/oily stains, for prevention of the staining /discoloration of the dishware and 5 plastic components of the dishwasher by highly colored components and the avoidance of lime soap deposits on the dishware.

Other uses

The phospholipase of the invention can be used to improve the filterability of an aqueous solution or slurry of carbohydrate origin by treating it with the phospholipase. This is

10 particularly applicable to a solution of slurry containing a starch hydrolyzate, especially a wheat starch hydrolyzate, since this tends to be difficult to filter and to give cloudy filtrates. The treatment can be done in analogy with EP 219,269 (CPC International).

Further, the phospholipase of the invention may be used for partial hydrolysis of phospholipids, particularly lecithin, to obtain improved phospholipid emulsifiers. This applica-

15 tion is further described in Ullmann's Encyclopedia of Industrial Chemistry (Publisher: VCH Weinheim (1996)), JP patent 2794574, and JP-B 6-087751.

Further, the phospholipase of the invention may be used in a process for the production of an animal feed which comprises mixing the phospholipase with feed substances and at least one phospholipid. This can be done in analogy with EP 743 017.

20 Even further the phospholipase of the invention can be used in a process for reducing the content of phospholipid in an edible oil, comprising treating the oil with the phospholipase so as to hydrolyze a major part of the phospholipid, and separating an aqueous phase containing the hydrolyzed phospholipid from the oil. This process is applicable to the purification of any edible oil which contains phospholipid, e.g. vegetable oil such as soy bean oil,

25 rape seed oil and sunflower oil. JP-A 2-153997, US 5264367.

The phospholipase can also be used for making cheese as described in WO 00/54601.

EXAMPLES

Materials and methods

30 Methods

Unless otherwise stated, DNA manipulations and transformations were performed using standard methods of molecular biology as described in Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.) "Current protocols in Molecular Biology", John Wiley and Sons,

35 1995; Harwood, C. R., and Cutting, S. M. (eds.) . Enzymes

Enzymes for DNA manipulations (e.g. restriction endonucleases, ligases etc.) are obtainable from New England Biolabs, Inc. and were used according to the manufacturer's instructions. Plasmids/vectors pT7Blue (Invitrogen, Netherlands)

Cloning

LA PCR™ in vitro Cloning Kit (TaKaRa) was used for cloning according to the manufacturer's instructions.

Microbial strains

E. coli JM109 (TOYOBO, Japan)

E.coli DB6507 (F,pnrF74::Tn5,seupE44, lacY1, ara-14,galK2, xyl-5, mtl-1, leuB6, proA2, hsdS20, recA13, rpsL20, thi-1, lambda-)

Media and reagents

Cove: 342.3 g/L Sucrose, 20 ml/L COVE salt solution, 10mM Acetamide, 30 g/L noble agar.

Cove-2: 30 g/L Sucrose, 20 ml/L COVE salt solution, 10mM, Acetamide, 30 g/L noble agar. Cove salt solution: per liter 26 g KCl, 26 g MgSO4-7aq, 76 g KH2PO4, 50ml Cove trace metals.

Cove trace metals: per liter 0.04 g NaB4O7-10aq, 0.4 g CuSO4-5aq, 1.2 g FeSO4- 7aq, 0.7 g MnSO4-aq, 0.7 g Na2MoO2-2aq, 0.7 g ZnSO4-7aq.

AMG trace metals: per liter 14.3 g ZnSO4-7aq, 2.5 g CuSO4-5aq, 0.5 g NiCI2, 13.8 g FeSO4, 8.5 g MnSO4, 3.0 g citric acid.

YPG: 4 g/L Yeast extract, 1 g/L KH2PO4, 0.5 g/L MgSO4-7aq, 5 g/L Glucose, pH 6.0.

STC: 0.8 M Sorbitol, 25 mM Tris pH 8, 25 mM CaCI2. STPC: 40 % PEG4000 in STC buffer. Cove top agarose: 342.3 g/L Sucrose, 20 ml/L COVE salt solution, 10mM

Acetamide, 10 g/L low melt agarose.

MLC : per liter 40 g glucose, 50 g soybean powder, 4 g citric acid, pH 5.0. MU-1: per liter 260 g maltodextrin, 3 g MgSO4-7aq, 6 g K2SO4, 5 g KH2PO4, 0.5 ml Trace metal solution for AMG (MU-1) Trace metal solution for AMG (MU-1) : per 100 ml 1.39 g FeSO4-7aq, 1.356 g

MnSO4-5aq, 0.68 g ZnCI2, 0.25 g CuSO4-5aq, 0.024 g NiCI2-6aq, 0.3 g Citric acid MT-O medium

Soybean powder 30 g/liter

Corn steep powder 5 g/liter

Yeast extract 1 g/liter

Peptone 5 g/liter

Glucose 10 g/liter

NH4NO3 2.5 g/liter

K2HPO4 4 g/liter

MgSO4 7 H2O 0.1 g/liter

Olive oil 2 ml/shake flask

Adeka Pluronic L-61 2 drops/shake flask

Deionized water is used. pH is adjusted to 6.5 before autoclaving. 100 ml substrate/shake flask (500 ml). Adeka Pluronic L-61 is an antifoam from Asahi Denka Kogyo K.K. (Japan). Lecithin plates

Substrate: 0.5 % L-alpha Phosphatidyl Choline 95% (AVANTI 441601). Emulsifier: 0.25% Cholic acid Buffer: 50mM acetate buffer pH5.5 10mM CaCl2 Agar Noble 2%

2% Brilliant Green 15ml/L

Example 1: Production of phospholipase

Zygoascus hellenicus CBS 4075 was grown for 4 days at 25°C on PDA slants (product of Difco with 1 % extra agar added). Cells were harvested in 0.1% Tween 20 water and 57 MT-O shake flasks were inoculated and incubated on a rotary shaker at 30°C for 3 days.

Cells and particulate matter were removed by centrifugation giving 4.4 liter of supernatant. The supernatant was concentrated by ultrafiltration, washed with ca. 2 volumes of deionized water and freezedried. About 40 g of freeze-dried powder was obtained.

Example 2: Purification of wild type Phospholipase

1-gram powder from Example 1 was dissolved in 100 ml water and ultra filtrated using Amicon filter (YM10 kDa cut off). After concentration to 10 ml, pH of the solution was adjusted to 8 and conductivity was 2 mSi. The sample was then applied on anion exchanger FFQ sepharose equilibrated with 50 mM Borate buffer pH 8. Bound enzyme was eluted using linear salt gradient using same buffer containing 1 M salt. Fractions containing protein were collected.

SDS-PAGE of the fractions showed a single band.

N-terminal of the blotted protein was determined. The sequence of the N-terminal was as shown in SEQ ID NO: 8.

The enzyme was qualitatively assayed to confirm phospholipase activity using Lecithin as substrate and NEFA-kit from Waco chemicals to determine free fatty acids released.

Example 3: Cloning and expression of lipase gene from Zygoascus hellenicus Cloning of phospholipase gene from Zygoascus hellenicus

A strain of Zygoascus hellenicus was used as a genomic DNA supplier. Cloning was carried out with adaptor PCR method using LA PCR™ in vitro Cloning Kit (TaKaRa)

PCR reactions on Zygoasucus hellenicus genomic DNA which was digested by Pst I and ligated to Pst I cassette of the kit was done with ZhelN-2 (SEQ ID NO: 3) designed from N- terminal amino acid sequence of purified protein.

Reaction components (20ng /μl of genomic DNA with cassette, 1 mM dNTP each, primer 2 nM each, 2.5mM MgCI2, 0.5 U/ μl in LA Taq polymerase in 1X buffer (TaKaRa, Japan)) were mixed and submitted to PCR under the following conditions.

Steps 1 to 3 were repeated 30 times. Amplified 640bp of the fragment was purified by GFX™ PCR DNA and Gel Band

Purification kit (Amersham Pharmacia Biotech) and sequenced.

In order to clone the missing part of the gene, genome walking was done. N-terminal part was obtained from the reaction mixture; 20 ng/μl of genomic DNA with BamH I cassette, 1 mM dNTP each, 2 nM, ZNr-2 primer, 2 nM, cassette primer, 2.5mM MgCI2, 0.5 U/ μl in LA Taq polymerase in 1X buffer (TaKaRa, Japan). ZNr-2: 5'-cttgtagagcagctggtaagatttc-3'

Steps 1 to 3 were repeated 30 times. C-terminal part was obtained by nested PCR with the primers ZC-1 and ZC-2 (SEQ ID NOS: 4 and 5) and genomic DNA with BamH I cassette. Reaction condition was same as above.

Obtained fragments were purified by GFX™ PCR DNA and Gel Band Purification kit (amersham pharmacia biotech) and sequenced with each primer which amplified the fragment.

The fidelity of LA-taq polymerase is not so good so in order to get the right sequence whole gene was amplified the primers Zhel-N (Bam) and Zhel-C (Sal) (SEQ ID NOS: 6 and 7).

Reaction components (6 ng /μl of genomic DNA, 250 mM dNTP each, primer 250 nM each, 0.05 U/ μl of Expand high fidelity polymerase in 1X buffer (Roche Diagnostics, Japan)) were mixed and submitted for PCR under the following conditions.

An amplified DNA fragment was gel-purified with GFX™ PCR DNA and Gel Band

Purification kit (Amersham Pharmacia Biotech) and ligated into a pT7Blue vector with ligation high (TOYOBO, Japan) . The ligation mixtures were transformed into E. coli JM109. The resultant plasmids, pTZhel-2, pTZhel-5, pTZhel-6, and pTZhel-7, were sequenced and all of them are identical. The sequence is defined as Zygoascus hellenicus phospholipase DNA sequence.

Expression of phospholipase gene in Aspergillus niger.

The phospholipase gene was digested from pTZhel-5 with BamH I and Xho I and ligated into the BamH I and Xhol sites in the Aspergillus expression cassette pMT2188 which has Aspergillus niger neutral amylase promoter, Aspergillus nidulans TPI leader sequences, Aspergillus niger glucoamylase terminator and Aspergillus nidulans amdS gene as a marker and Saccharomyces cerevisiae URA3 gene as a marker for a plasmid construction. The ligation mixture was transformed E.coli 6507 by electroporation and the resultant plasmid was pZPL-8. A strain of Aspergillus niger was inoculated to 100 ml of YPG medium and incubated for16 hrs at 32°C at 120 rpm. Pellets were collected and washed with 0.6 M KCl, and resuspended 20 ml 0.6 M KCl containing a commercial β-glucanase product (Glucanex, product of Novo Nordisk A/S) at the concentration of 30 μl/ml. Cultures were incubated at 32°C at 60 rpm until protoplasts formed, then washed with STC buffer twice. The protoplasts were counted with a hematometer and resuspended in an 8:2:0.1 solution of STC:STPC:DMSO to a final concentration of 2.5x10e7 protoplasts/ml. About 3 μg of DNA was added to 100 μl of protoplasts solution, mixed gently and incubated on ice for 30 min. One ml of SPTC was added and incubated 30 min at 37°C. After the addition of 10 ml of 50°C Cove top agarose, the reaction was poured onto Cove agar plate. Transformation plates were incubated at 32°C for 5 days. pZPL-8 was transformed into the strain of Aspergillus niger. The selected transfor- mants were inoculated in 100 ml of MLC media and cultivated at 32°C for 2 day. 5 ml of grown cell in MLC medium was inoculated to 100 ml of MU-1 medium and cultivated at 32°C for 3 days. The supernatant was obtained by centrifugation. The phospholipase activity was checked by a lecitin plate whose composition is described above.

The substrate and the emulsifier were dissolved together in appropriate amount of D.W and autoclaved briefly (120°C for 5min). The solution was stirred until the small particles had disappeared. Afterward it was subjected to hiscotron for 5min. It was mixed well with other components and 25ml aliquots were poured into Falcon 1004 plates.

Supernatant of transformants showed phospholipase activity with green halo on the assay plate.

Original (for SUBMISSION) - printed on 18.09.2001 09:06:24 AM

- 0-1 Form - PCT/RO/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 . 03 .2001)

0-2 International Application No.

P C T/D K 0 1 / O O β O O

0-3 Applicant's or agent's file reference 10078-WO

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 25-28

1-3 Identification of Deposit

1-3-1 Name of depositary institution DSMZ-Deutsc e Sammlung von

Mikroorganismen und Zellkulturen GmbH

1-3-2 Address of depositary institution Mascheroder Weg lb , D-38124

Braunschweig , Germany

1-3-3 Date of deposit 10 August 2000 (10 . 08 . 2000) 1-3-4 Accession Number DSMZ 13648

1-4 Additional Indications NONE

1-5 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

1. A phospholipase which is: a) a polypeptide encoded by the phospholipase encoding part of the DNA sequence cloned into a plasmid present in Escherichia coli deposit number DSM 13648, or 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 (a) or (b) which: i) has at least 50 % identity 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, d) a polypeptide which is encoded by a nucleic acid sequence which hybridizes at 55°C 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 nu- cleotides.

2. The phospholipase of claim 1 which is native to a strain of Zygoascus, particularly Z. hellenicus, more particularly strain CBS 4075.

3. A nucleic acid sequence comprising a nucleic acid sequence which encodes the phospholipase of claim 1 or 2.

4. A nucleic acid sequence which comprises: a) the partial DNA sequence encoding a mature phospholipase cloned into a plasmid present in Escherichia coli DSM 13648, b) the partial DNA sequence encoding a mature phospholipase shown in SEQ ID

NO: 1 , c) an analogue of the sequence defined in a) or b) which encodes a phospholipase and i) has at least 60 % identity with said DNA sequence, or ii) hybridizes at 55°C 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 a), b) or c).

5. A nucleic acid construct comprising the nucleic acid sequence of claim 3 or 4 opera- bly linked to one or more control sequences capable of directing the expression of the phospholipase in a suitable expression host.

6. A recombinant expression vector comprising the nucleic acid construct of claim 5, a promoter, and transcriptional and translational stop signals.

7. A recombinant host cell comprising the nucleic acid construct of claim 6.

8. A method for producing a phospholipase comprising cultivating the host cell of claim 7 under conditions conducive to production of the phospholipase, and recovering the phospholipase.

9. A method for preparing a dough or a baked product made from the dough, comprising adding the phospholipase of claim 1 to the dough.

10. A dough composition comprising the phospholipase of claim 1.

11. A detergent composition comprising a surfactant and the phospholipase of claim 1.

12. A process for reducing the content of phosphorus in a vegetable oil, comprising con- tacting the oil with the phospholipase of claim 1 in the presence of water, and then separating an aqueous phase from the oil.