Phospholipase C

Abstract

The present invention relates to a polypeptide having phospholipase C activity, selected from the group consisting of i. a polypeptide comprising a mature polypeptide sequence of SEQ ID NO: 2; ii. a polypeptide that has least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the mature polypeptide sequence of SEQ ID NO: 2; iii. a polypeptide encoded by a nucleic acid that hybridizes under medium stringency, preferably under high stringency conditions to the complementary strand of the mature polypeptide coding sequence of SEQ ID NO:1; iv. a polypeptide encoded by a nucleic acid that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the mature polypeptide coding sequence of SEQ ID NO: 1. A process for degumming a vegetable oil comprising contacting a vegetable oil comprising phospholipids with a polypeptide having phospholipase C activity of the invention or a composition of the invention, wherein phospholipids are hydrolyzed into diacylglycerol and phosphate ester and/or phosphate, separating the phosphate ester and/or phosphate from the vegetable oil wherein a degummed vegetable oil is obtained.

Claims

1. A process for hydrolysing one or more phospholipids comprising: a) separating the one or more phospholipids from an oil; b) incubating the one or more phospholipids with a polypeptide having phospholipase C activity selected from the group consisting of: i) a polypeptide comprising a mature polypeptide sequence of SEQ ID NO: 2; ii) a polypeptide that has at least 85% sequence identity to the mature polypeptide sequence of SEQ ID NO: 2; and iii) a polypeptide encoded by a nucleic acid that has at least 85% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1, wherein the one or more phospholipids are hydrolysed.

2. The process according to claim 1, wherein the one or more phospholipids comprise phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine and/or phosphatidic acid.

3. A process for degumming a vegetable oil comprising: a) contacting a vegetable oil comprising one or more phospholipids with a polypeptide having phospholipase C activity or a composition thereof, wherein the one or more phospholipids are hydrolysed into diacylglycerol and phosphate ester and/or phosphate, and wherein the polypeptide having phospholipase C activity is selected from the group consisting of: i) a polypeptide comprising a mature polypeptide sequence of SEQ ID NO: 2; ii) a polypeptide that has at least 85% sequence identity to the mature polypeptide sequence of SEQ ID NO: 2; and iii) a polypeptide encoded by a nucleic acid that has at least 85% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1; and b) separating the phosphate ester and/or phosphate from the vegetable oil wherein a degummed vegetable oil is obtained.

4. The process for degumming according to claim 3, wherein the one or more phospholipids are hydrolysed at a pH value of between 3 and 9.

5. The process according to claim 3, wherein the one or more phospholipids phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine and/or phosphatidic acid are hydrolysed.

Description

FIGURES

(1) FIG. 1: Schematic representation of the vector pGBTOP16 used for cloning the phospholipase C gene.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

(2) The present invention relates to a polypeptide having phospholipase C activity, selected from the group consisting of i. a polypeptide comprising a mature polypeptide sequence of SEQ ID NO: 2; ii. a polypeptide that has least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the mature polypeptide sequence of SEQ ID NO: 2; iii. a polypeptide encoded by a nucleic acid that hybridizes under medium stringency, preferably under high stringency conditions to the complementary strand of the mature polypeptide coding sequence of SEQ ID NO:1; iv. a polypeptide encoded by a nucleic acid that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the mature polypeptide coding sequence of SEQ ID NO: 1.

(3) Surprisingly, it was found that a polypeptide as defined herein having phospholipase C activity comprises activity towards all of the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidic acid (PA). It is advantageous in oil degumming processes that a single phospholipase can be used to hydrolyse the major phospholipids instead of using a mixture of different phospholipases, such as phospholipase C, phosphatidyl-inositol specific (PI-PLC), or phospholipase A, which have activity on one or a selection of the major phospholipids PC, PE, PI or PA.

(4) A polypeptide as disclosed herein has a pH optimum of about 4.5 to 5. For instance a pH optimum of a polypeptide having phospholipase C activity as disclosed herein may be determined by measuring phospholipase C activity in an aqueous solution comprising 100 mM acetate, 1% Triton X-100 and 1 mM ZnSO.sub.4 with p-nitrophenyl phosphorylcholine (pNP-PC) as a substrate at 37 C.

(5) Surprisingly, it was found that a polypeptide having phospholipase C activity as disclosed herein, more efficiently degraded phospholipids such as phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidic acid (PA) in a neutral environment than in a more acid environment in a high oil/low water composition. A high oil/low water composition comprises less than 10, 8, 6, or 4 wt % of water relative to oil.

(6) A polypeptide as disclosed herein may be an isolated, substantially pure, pure, recombinant, synthetic or variant polypeptide or a non-naturally occurring polypeptide.

(7) A polypeptide having phospholipase C activity according to the present invention is advantageously derived from a Hypocrea sp., eg. Hypocrea virens. The wording derived or derivable from, with respect to the origin of a polypeptide as disclosed herein, means that when carrying out a BLAST search with a polypeptide according to the present invention, the polypeptide according to the present invention may be derivable from a natural source, such as a microbial cell, of which an endogenous polypeptide shows the highest percentage homology or identity with the polypeptide as disclosed herein.

(8) In one embodiment, a polypeptide of the invention having phospholipase C activity is not a native polypeptide of Hypocrea virens containing a native leader sequence. For instance, a polypeptide of the invention having phospholipase C activity is not the complete amino acid sequence of SEQ ID NO: 2. For instance, a polypeptide of the invention having phospholipase C activity is not the amino acid sequence consisting of amino acids 1 to 643 of SEQ ID NO: 2.

(9) A polypeptide having phospholipase C activity (EC 3.1.4.3) as disclosed herein is an enzyme that may hydrolyze phospholipids into diacylglyceride (DAG) and a phosphate from phosphatidic acid (PA), or DAG and a phosphate ester (from PC, PE and PI). Surprisingly, it was found that a polypeptide having phospholipase C activity according to the present invention has activity to the major phospholipids phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, and phosphatidic acid. An advantage of a polypeptide having activity to the major phospholipids is that only one phospholipase enzyme may be needed to hydrolyze all phospholipids into diglyceride and phosphate or phosphate ester, such as in a vegetable oil degumming process for efficient removal of phospholipids. A phospholipase as disclosed herein may have phosphatase activity E.C. 3.1.3. A phosphatase catalyzes the conversion of a phosphate ester, for instance the conversion of phosphate esters PC PE and PI into phosphate and choline, ethanol amine and inositol, respectively.

(10) A polypeptide of SEQ ID NO: 2 comprises a leader sequence for secretion of the polypeptide outside cell. A leader sequence in SEQ ID NO: 2 may comprise amino acids 1 to 15, or 1 to 16, or 1 to 17, or 1 to 18 or 1 to 19, wherein the methionine (M) at position 1 is counted as 1.

(11) A mature polypeptide sequence of SEQ ID NO: 2 as disclosed herein may comprise the amino acids from position 16, 17, 18, 19, or 20 to position 638, 639, 640, 641, 642 or 643 of SEQ ID NO: 2 wherein the methionine (M) at position 1 is counted as 1. For example a mature polypeptide of SEQ ID NO: 2 may comprise the amino acids from position 19 to position 643 of SEQ ID NO: 2, or is amino acids 19 to 643 of SEQ ID NO: 2, wherein the methionine (M) at position 1 is counted as 1. A polypeptide that has least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the mature polypeptide sequence of SEQ ID NO: 2, may be a polypeptide that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to amino acids 19 to 643 of SEQ ID NO: 2.

(12) A polypeptide according to the present disclosure may advantageously be an isolated, substantially pure, pure, recombinant, synthetic or variant polypeptide of the polypeptide as defined herein. A polypeptide as disclosed herein may be purified. Purification of protein is known to a skilled person in the art, and may for instance comprise steps as separating proteins from cells or cell fractions, for instance by centrifugation, ammonium sulphate precipitation, ultracentrifugation, chromatography, or filtration, or ultrafiltration.

(13) In one aspect the present disclosure relates to a composition comprising a polypeptide as disclosed herein. A composition as disclosed herein, may comprise a carrier, an excipient, an auxiliary enzyme, or other compounds. Typically a composition, or a formulation, comprises a compound with which a polypeptide having phospholipase C activity may be formulated. An excipient as used herein is an inactive substance formulated alongside with a polypeptide as disclosed herein, for instance sucrose or lactose, glycerol, sorbitol or sodium chloride. A composition comprising a polypeptide as disclosed herein may be a liquid composition or a solid composition. A liquid composition, typically an aqueous solution, usually comprises water. When formulated as a liquid composition, the composition usually comprises components that lower the water activity, such as glycerol, sorbitol or sodium chloride (NaCl). A solid composition comprising a polypeptide as disclosed herein may comprise a granulate comprising the enzyme or the composition comprises an encapsulated polypeptide in liquid matrices like liposomes or gels like alginate or carrageenans, or (synthetic.resins or silicas). There are many techniques known in the art to encapsulate or granulate a polypeptide or enzyme (see for instance G. M. H. Meesters, Encapsulation of Enzymes and Peptides, Chapter 9, in N. J. Zuidam and V. A. Nedovid (eds.) Encapsulation Technologies for Active Food Ingredients and food processing 2010).

(14) A composition as disclosed herein may also comprise a carrier comprising a polypeptide as disclosed herein. A polypeptide as disclosed herein may be bound or immobilized to a carrier by known technologies in the art, for instance by immobilizing the polypeptide on a carrier such as alginate or carrageenan. A composition as disclosed herein may also comprise an auxiliary enzyme, for instance a phospholipase A, or a PI-PLC. Alternatively, a composition as disclosed herein may comprise other enzymes that may be beneficial to enzyme assisted oil-degumming or refining. For instance a composition as disclosed herein may comprise one or more further enzyme(s) such as proteases, a chlorophyllases, pheophytinases, carbohydrases, for instance cell wall degrading enzymes such as cellulases, hemicellulases, pectinases and/or -glucosidases, and/or lipases.

(15) A composition comprising a polypeptide having phospholipase C activity as disclosed herein may also be a fermentation broth comprising the polypeptide. A composition may comprise a polypeptide having phospholipase C activity wherein the polypeptide is bound to cells or cell material. Alternatively, cells or cell material have been removed from the fermentation broth by centrifugation. Optionally, cells are killed for instance by a heating step.

(16) The present invention also relates to a process for preparing a composition comprising a polypeptide as disclosed herein, which may comprise spray drying a fermentation medium comprising the polypeptide, or granulating, or encapsulating a polypeptide as disclosed herein, and preparing the composition. Spray drying is known to a skilled person and usually comprises producing a dry powder from a liquid or slurry comprising a polypeptide as disclosed herein by rapidly drying with hot gas.

(17) In another embodiment the present invention relates to a nucleic acid encoding a polypeptide having phospholipase C activity as disclosed herein, which has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 or to the mature polypeptide encoding sequence of SEQ ID NO: 1.

(18) A nucleic acid of the present disclosure may be an isolated, substantially pure, pure, recombinant, synthetic or variant nucleic acid of the nucleic acid or a non-naturally occurring nucleic acid. A polynucleotide sequence as disclosed herein may comprise SEQ ID NO: 1, or may comprise the mature polypeptide encoding sequence of SEQ ID NO:1.

(19) In one other embodiment of the present invention a nucleic acid is disclosed that is an isolated, substantially pure, pure, recombinant, synthetic or variant nucleic acid of the nucleic acid of SEQ ID NO: 1. A variant nucleic acid sequence may for instance have at least 80% sequence identity to SEQ ID NO:1.

(20) In another aspect, the present invention relates to an expression vector comprising a nucleic acid as disclosed herein operably linked to one or more control sequence(s) that direct expression of the polypeptide in an expression host cell.

(21) An expression vector may be obtained for instance by inserting a nucleic acid of the present invention into an empty construct or vector. There are several ways of inserting a nucleic acid into a nucleic acid construct or an expression vector which are known to a skilled person in the art, see for instance Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, N.Y., 2001. It may be desirable to manipulate a nucleic acid encoding a polypeptide of the present invention with suitable control sequences. For instance the one or more control sequence(s) in an expression vector as disclosed herein may comprise a promoter sequence, a terminator sequence, and/or a leader sequence.

(22) A promoter may be any appropriate promoter sequence suitable for a eukaryotic or prokaryotic host cell, which shows transcriptional activity, including mutant, truncated, and hybrid promoters, and may be obtained from polynucleotides encoding extracellular or intracellular polypeptides either endogenous (native) or heterologous (foreign) to the cell. The promoter may be a constitutive or inducible promoter. Preferably, the promoter is an inducible promoter, for instance a starch inducible promoter. Promoters suitable in filamentous fungi are promoters which may be selected from the group, which includes but is not limited to promoters obtained from the polynucleotides encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus gpdA promoter, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger or A. awamori glucoamylase (glaA), A. niger or A. awamori endoxylanase (xInA) or beta-xylosidase (xInD), T. reesei cellobiohydrolase I (CBHI), R. miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, A. nidulans acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a hybrid of the promoters from the polynucleotides encoding A. niger neutral alpha-amylase and A. oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof.

(23) Any terminator which is functional in a cell as disclosed herein may be used, which are known to a skilled person in the art. Examples of suitable terminator sequences in filamentous fungi include terminator sequences of a filamentous fungal gene, such as from Aspergillus genes, for instance from the gene A. oryzae TAKA amylase, the genes encoding A. niger glucoamylase (glaA), A. nidulans anthranilate synthase, A. niger alpha-glucosidase, trpC and/or Fusarium oxysporum trypsin-like protease.

(24) In another embodiment the present invention relates to a recombinant host cell comprising a nucleic acid or an expression vector as disclosed herein. A suitable host cell may be a mammalian, insect, plant, fungal, or algal cell, or a bacterial cell. A suitable bacterial cell may be from the genera Bacillus, Streptomyces or Pseudomonas, for instance from the species B. amyloliquefaciens, B. licheformis, S. coelicolor, or P. putida.

(25) A suitable host cell may be a fungal cell, for instance from the genus Acremonium, Aspergillus, Chrysosporium, Fusarium, Myceliophthora, Penicillium, Rasamsonia, Talaromyces, Thielavia, Trichoderma, Saccharomyces, Kluyveromyces, Pichia, for instance Aspergillus niger, Aspergillus awamori, Aspergillus foetidus, A. oryzae, A. sojae, Talaromyces emersonii, Rasamsonia emersonii Chrysosporium lucknowense, Fusarium oxysporum, Myceliophthora thermophila, Thielavia terrestris or Trichoderma reesei or, Saccharomyces cerevisiae, Kluyveromyces lactis, Pichia pastoris. Preferably, a recombinant or transgenic host cell for expression of a phospholipase C as disclosed herein is an Aspergillus niger.

(26) The host cell may be genetically modified with a nucleic acid construct or expression vector as disclosed herein with standard techniques known in the art, such as electroporation, protoplast transformation or conjugation for instance as disclosed in Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, N.Y., 2001.

(27) In one aspect the present invention relates to a process for the production of a polypeptide as disclosed herein comprising cultivating a recombinant host cell as disclosed herein in a suitable fermentation medium under conditions conducive to the production of the polypeptide and producing the polypeptide. A skilled person in the art understands how to perform a process for the production of a polypeptide as disclosed herein depending on a host cell used, such as pH, temperature and composition of a fermentation medium. A suitable fermentation medium usually comprises nutrients such a nitrogen, a carbon source and other essential elements known to a skilled person in the art for cultivating a particular host cell.

(28) Host cells can be cultivated in shake flasks, or in fermenters having a volume of 0.5 or 1 liter or larger to 10 to 100 or more cubic meters. Cultivation may be performed aerobically or anaerobically depending on the requirements of a host cell.

(29) A process for preparing a polypeptide as disclosed herein may further comprise recovering the polypeptide from the fermentation medium. Recovering the polypeptide may comprise isolating the polypeptide. Recovering may be performed by known methods in the art for instance recovering may comprise filtration, ultrafiltration, microfiltration, centrifugation or chromatography.

(30) In another aspect the present invention relates to a process for hydrolysing phospholipids comprising incubating the phospholipids with a polypeptide having phospholipase C activity as disclosed herein or a composition as disclosed herein, wherein the phospholipids are hydrolysed. Phospholipids that may be hydrolysed include phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidic acid, and/or phosphatidyl serine. Phospholipids may be derived from a vegetable oil or a fat, for instance an animal derived fat such a dairy fat. Surprisingly, it was found that at least part of the phospholipids phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidic acid and phosphatidyl serine were hydrolysed by a polypeptide having phospholipase C activity as disclosed herein.

(31) A process for hydrolysing phospholipids may comprise a step wherein phospholipids are separated from an oil, for instance a vegetable oil, or from a fat, for instance a dairy fat, for instance by water degumming, resulting in phospholipid-containing gums and water-degummed oil; incubating the phospholipid-containing gums with a polypeptide having phospholipase C activity as disclosed herein, wherein phospholipids are hydrolysed. A neutral oil fraction is usually obtained upon hydrolysis of the phospholipids and can be separated from the phosphor-containing fraction. The neutral oil can be then be added to the water degummed oil, which increases the yield of the oil.

(32) Incubating phospholipids with a polypeptide having phospholipase C activity may be performed at any suitable pH and temperature. A suitable pH may for instance be a pH of between 3 and 9, or a pH of between 4 and 8, or a pH of between 5 and 7, or a pH of between 6 and 8. A suitable temperature may be a temperature of between 20 and 80 C., for instance between 30 and 70 C., such as between 40 and 60 C. or between 45 and 55 C.

(33) In another aspect the present invention relates to a process for degumming a vegetable oil comprising contacting a vegetable oil comprising one or more phospholipids with a polypeptide having phospholipase C activity as disclosed herein or a composition as disclosed herein, wherein the one or more phospholipids are hydrolysed into diacylglycerol and phosphate ester and/or phosphate, separating the phosphate ester and/or phosphate from the vegetable oil wherein a degummed vegetable oil is obtained. In a process for degumming as disclosed herein hydrolysing phospholipids means that at least part of the phospholipids is hydrolysed. For instance, at least a part of the phospholipids phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidic acid and/or phosphatidyl serine is hydrolysed.

(34) Degumming a vegetable oil in a process as disclosed herein comprises reducing the phosphorus content in the vegetable oil. Advantageously, the atomic phosphorus content is reduced to less than 50 ppm P, or less than 20 ppm P or less than 10, or less than 5 ppm P, or less than 2 ppm P. The phosphorus content can be measured by standard ICP methods (inductively coupled plasma).

(35) A vegetable oil in a process according to the present disclosure may be a crude oil or a previously degummed oil by other means, for instance by water degumming. A vegetable oil may also be obtained by pressing oil seed (pressed oil or expeller), by hexane extraction of oil seed. A vegetable oil may for instance be canola oil, castor oil, coconut oil, corn oil, cotton oil palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, soybean oil or sunflower oil.

(36) A process for degumming a vegetable oil as disclosed herein may further comprise a step of separating the degummed vegetable oil from gums. Such separating may for instance be achieved by centrifugation.

(37) Contacting a vegetable oil comprising phospholipids with a polypeptide having phospholipase C activity may be performed in any suitable way. A polypeptide having phospholipase C activity may be dissolved in an aqueous medium and brought into contact with the vegetable oil by mixing or stirring, for instance by high-shear mixing.

(38) A process for degumming a vegetable oil may be carried out at a pH of between 3 and 9, or a pH between 4 and 8, or a pH between 5 and 7, or a pH between 4 and 6, or a pH of between 3 and 7. The pH can be adjusted by known methods in the art for instance by the addition of acids, such as citric or phosphoric acid, optionally subsequently or simultaneously adding caustic such as sodium hydroxide. Advantageously phospholipids are hydrolysed at an acid pH value, since when phospholipids are hydrated they are more easily hydrolysed.

(39) The temperature at which a process for degumming is carried out may be between 20 and 90 C., or between 25 and 80 C., such as between 30 and 70 C., or between 40 and 60 C., for instance between 45 and 55 C., for instance at a temperature of between 50 and 90 C. It was found advantageous to carry out a process for degumming at an elevated temperature, because the removal of gums rich in phosphorous-containing compounds by centrifugation is mostly done at elevated temperatures, such as 80 C. If the enzyme-assisted hydrolysis of phospholipids is performed at elevated temperature such as between 70 and 90 C., the temperature of the oil does not need to be lowered after hexane extraction or pressing and before enzyme-assisted hydrolysis.

(40) In one aspect the present invention relates to a degummed vegetable oil obtainable by a process as disclosed herein.

(41) The following examples illustrate the invention.

EXAMPLES

Materials and Methods

Example 1. Cloning and Expression of Phospholipase C

(42) A polypeptide of Hypocrea virens of SEQ ID NO: 2, (L061 sequence) comprises a signal sequence of 18 amino acids for efficient secretion in Hypocrea virens, and a deduced mature protein sequence of 625 amino acids.

(43) A codon-adapted DNA sequence for expression of the protein in Aspergillus niger was designed containing additional restriction sites for subcloning in an Aspergillus expression vector. Codon adaptation was performed as described in WO2008/000632. The codon optimized DNA sequence for expression of the gene encoding the L061 protein of SEQ ID NO: 2 in A. niger is shown in SEQ ID NO: 1.

(44) The translational initiation sequence of the glucoamylase glaA promoter was modified into 5-CACCGTCAAA ATG-3 (SEQ ID NO:3) and an optimal translational termination sequence 5-TAAA-3 was used in the generation of the expression constructs (as also detailed in WO2006/077258). A DNA fragment (SEQ ID NO: 4), containing a.o. part of the glucoamylase promoter and the PLC encoding genes, was synthesized completely, purified and digested with EcoRI and Pad.

(45) The pGBTOP-16 vector (FIG. 1) was linearized by EcoRI/PacI digestion and the linearized vector fragment was subsequently purified by gel-extraction. The DNA fragments were cloned into the pGBTOP-16 vector and the resulting vector was named pGBTOPL061. Subsequently, A. niger GBA 306 was transformed with pGBTOPL061 in a co-transformation protocol with pGBAAS-4, with strain and methods as described in WO 2011/009700 and references therein, and selected on acetamide containing media and colony purified according to standard procedures. Transformation and selection was performed as described in WO 98/46772 and WO 99/32617. Strains containing the L061 gene were selected via PCR with primers amplifying the introduced L061 gene to verify presence of the pGBTOPL061 expression cassette. A single transformant expressing NBL-L061 was selected, and further replica-plated to obtain a single strain inoculum.

Example 2. Fermentation of A. niger NBL-L061 Strain

(46) Fresh A. niger NBL-L061 spores were prepared and used for generating sample material by cultivation of the strain in 24 deep well plates (Axygen, Union City, USA) containing 3 ml fermentation medium 2 (15% w/v maltose, 6% w/v bacto-soytone, 1.5% w/v (NH.sub.4).sub.2SO.sub.4, 0.1% w/v NaH.sub.2PO.sub.4.H.sub.2O, 0.1% w/v MgSO.sub.4.7H.sub.2O, 0.1% w/v L-arginine, 8 w/v Tween-80, 2 w/v Basildon, 2% w/v MES, pH 5.1). The 24 deep well plates were covered with a Breathseal (Greiner bio-one, Frickenhausen, Germany) and a lid. After 6 days of growth at 34 C., 550 rpm and 80% humidity in a Microton incubator shaker (Infors AG, Bottmingen, Switzerland) 1.5 mL samples were taken, the mycelium was separated from the supernatant by centrifugation for 30 min at 4000 g and the supernatants were stored at 20 C. until further analyses.

Example 3. Activity Determination of Phospholipase L061

(47) 3.1. Phospholipase C (PLC) Activity Assay (for Screening)

(48) The PLC activity of the samples L061 A5 and C5 was determined using the chromogenic substrate p-nitrophenyl phosphorylcholine (pNP-PC). The substrate solution consisted of 10 mM pNP-PC (Sigma N5879, Zwijndrecht, the Netherlands), 100 mM acetate buffer

(49) pH 5.0, 1% Triton X-100 and 1 mM ZnSO.sub.4. A mixture of 20 L sample and 180 L substrate solution was incubated at 37 C. for 60 min. The reaction was stopped by adding 100 L reaction mixture to 100 L stop reagent containing 1 M TRIS and 50 mM EDTA adjusted to pH 10 with 2 M NaOH. A blank was made by adding the stop reagent before the enzyme sample. The optical density (OD) of samples and blanks were measured at 405 nm.

(50) Calibration was performed by preparing pNP solutions of respectively 0-0.5-1.0-2.0-2.9-4.0 mM in above mentioned buffer. 20 L of each standard solution was mixed with 180 L substrate and 100 L of the mixture was added to 100 L stop reagent. The OD of each solution was measured at 405 nm. By using linear regression, the slope of the calibration line was calculated.

(51) Activity was calculated by using the following formula:

(52) $U\text{/}\mathrm{mL}=\frac{\mathrm{Abs}\mathrm{Df}}{t*\mathrm{slope}}$

(53) Abs=(A.sub.sampleA.sub.blank)

(54) Df=dilution factor of sample

(55) slope=slope of p-nitro-phenol calibration curve (mL/mol)

(56) t=incubation time assay (60 min)

(57) One U is defined as the amount of enzyme that liberates 1 mol p-nitrophenol per minute under the conditions of the test (pH 5, 37 C.).

(58) 3.2. Phospholipase C (PLC) Activity Assay (for General Activity Measurement)

(59) The PLC activity of samples L061 SF1, SF2 and SF3 was determined as described under 3.1. with the exception that an acetate buffer with 0.2% Triton X-100 was used and a mixture of 40 L sample* and 960 L substrate solution was incubated at 37 C. for 30 min.

(60) *The sample had been diluted in 100 mM acetate buffer pH 5.0 with 0.2% triton X-100 and 1.0 mM Zinc sulfate. After centrifugation, the supernatant was used for the activity assay. The sample was diluted in such a way that the Abs is between 0.1 and 1.0.

(61) Accordingly, calibration was performed by preparing pNP solutions in 1000 l and incubation was performed for 30 min.

(62) 3.3. Protein Determination

(63) Protein content was measured according the Coomassie Plus protein kit of Pierce (art. no. 23236).

(64) See also internal method of analysis 2459Universal Bradford protein assay using Coomassie Plus assay reagent, manual method.

(65) Results of the PLC activity assays, protein determinations and specific activity are given in Table 2a and Table 2b

(66) TABLE-US-00001 TABLE 1 Phospholipase C activity of different screening samples of Hypocrea virens gene codon optimized for and expressed in Aspergillus niger. Enzyme/g Sample OD Protein Activity S.A. lecithin* ID 405 nm mg/ml U/ml U/mg mg/g U/g L061 A5 2.325 0.34 0.68 2.0 0.17 0.34 L061 C5 2.899 0.12 0.87 7.2 0.06 0.44

(67) TABLE-US-00002 TABLE 2 Phospholipase C activity of different samples of the Hypocrea virens gene codon optimized and expressed in Aspergillus niger. Protein Activity S.A. Sample ID Mg/ml U/ml U/mg L061 SF1 conc 3.1 17.1 5.5 L061 SF2 pure sup 0.06 L061 SF3 wh b* 0.12 1.0* 8 *Determined with SDS/PAGE, so only BS-PLC content

(68) SF2 pure sup: supernatant from a shake flask production; SF1 conc is a concentrated version thereof; SF3 wh b is a third shake flask production, whole broth (without separation of the cell material from the supernatant.). Shake flask productions were done in 100 mL flasks with about 50 gram of material in the same way as described for the small scale sample.

Example 4. Determination of pH and Temperature Profile

(69) The pH profile of phospholipase L061 was determined in a solution of 10 mM pNP-PC, 30 mM acetic acid, 30 mM MOPS, 30 mM MES, 0.2% triton X-100 and 1 mM ZnSO.sub.4 adjusted with NaOH to a pH ranging from 4 to 7.5. Subsequently the same procedure as described in Example 3 was followed. A shake-flask-produced enzyme sample with an activity of 1.0 U/mL was used.

(70) The temperature profile phospholipase L061 was determined using the method of Example 3 at different temperatures, using an enzyme sample prepared by a shake flask fermentation with an activity of 0.06 U/mL.

(71) The results in Table 3 and Table 4 show that the mature phospholipase L061 according to SEQ ID NO: 2 has a pH optimum of about 5.0 and a temperature optimum of about 55 C.

(72) TABLE-US-00003 TABLE 3 pH profile of phospholipase L061 (pH optimum is set at 100%) relative activity pH (%) 4.0 78.7 4.5 96.1 5.0 100.0 5.5 70.7 6.0 27.5 6.5 7.1 7.0 3.8 7.5 4.0

(73) TABLE-US-00004 TABLE 4 Temperature profile of phospholipase L061 (temperature optimum is set at 100%) Temperature relative activity ( C.) (%) 20.1 14.3 30.1 33.4 37.2 56.0 43.9 80.6 47.8 93.7 52.0 96.4 55.0 100.0 60.1 73.9 65.0 29.7 69.8 7.0

Example 5. Hydrolysis of Phospholipids in a Dispersion of De-Oiled Lecithin in Water at pH 5 and 6 and 60 C.

(74) 5.1. General Method:

(75) 10 wt % of de-oiled lecithin [DOL, Lecisoya P97IP, Novastell, France] was dispersed in a buffer, a 10 mM citrate buffer for pH 4 and 10 mM phosphate buffer for pH 6, using an Silverson L4RT dispersing device, room temperature 3 minutes 8000 rpm (80% of maximum). This was brought to 37 C. and enzyme was added in 3, 4 or 5 wt %, the amount depending on the protein content. When the supernatant contained more than 1.0 mg protein/mL, only 3 wt % of the material was added to the DOL dispersion, for a level between 0.5 and 1.0 mg/mL 4 wt % was added, for a level below 0.5 mg/mL 5 wt % was added.

(76) This dispersion was stirred at 37 C. or 60 C. for 24 hrs. After 4 and 24 hours a sample was taken. The sample was immediately heat treated to inactivate the enzyme by holding the sample for 5 minutes in a boiling water bath. Subsequently the samples were kept frozen until analysis by .sup.31P NMR.

(77) 5.2. .sup.31P NMR Method

(78) For .sup.31P NMR 10 L of 10% DOL dispersion was dispersed in 1 mL of an aqueous solvent containing demineralized water with 10% deuterium oxide (D.sub.2O, Cambridge Isotope Laboratories, DLM-4), 25 mg/mL deoxycholic acid (Sigma D2510), 5.84 mg/mL EDTA di Na (Titriplex III, Merck 108418), and 5.45 mg/mL TRIS base (Tris(hydroxymethyl) aminomethane, Merck 108387), of which the pH was adjusted to pH 9 using 4N KOH and to which 2 mg/mL TIP internal standard (tri-isopropylphosphate, Aldrich 554669) (accurately weighed) was added.

(79) All samples were measured in a Bruker 400 MHz AvanceIII NMR spectrometer with a Prodigy BBO probe. The temperature of the probe head was set at 300K.

(80) The measurement for quantification was performed with semi-quantitative parameters: 128 scans, 90 pulse, D1=5 sec. Values are reported in mol/g of dry weight (DOL) of the sample.

(81) 5.3. Incubation of L061 in a Dispersion of De-Oiled Lecithin at 60 C.

(82) 5 wt % of supernatant from sample L061 (Hypocrea virens; sample A5) was added to a DOL dispersion as described above, at pH 5 and 6. The DOL dispersion obtained after incubation for 24 hours and after inactivation of the enzyme was analysed by .sup.31P NMR. The .sup.31P NMR results are given in the following table, compared to the reference DOL dispersion kept for 24 hr at 60 C. at the same pH, in mol/g and in mol % (free phosphate levels excluded from mol % calculations).

(83) TABLE-US-00005 TABLE 5 Composition of de-oiled lecithin (DOL) incubated with L061 phospholipase at pH 5 and 6 at 60 C. for 24 hr. pH 5 and 6, 60 C. PL concentration PL amount in mol/g in mol fraction pH 5 pH 6 pH 5 pH 6 abbr Ref L061 Ref L061 Ref L061 Ref L061 Phosphatidyl choline PC 0.83 0.03 0.70 0.03 0.39 0.06 0.38 0.04 Choline phosphate C-P 0.01 0.19 0.01 0.29 0.00 0.34 0.00 0.40 Phosphatidyl PE 0.61 0.09 0.53 0.09 0.28 0.17 0.28 0.12 ethanolamine Ethanolamine E-P 0.21 0.08 0.01 0.09 0.00 0.13 0.00 0.12 phosphate Phosphatidyl inositol PI 0.50 0.01 0.43 0.01 0.23 0.03 0.23 0.02 Inositol phosphate IP 0.00 0.14 0.00 0.19 0.00 0.24 0.00 0.27 Phosphatidic acid PA 0.21 0.02 0.19 0.03 0.10 0.03 0.10 0.04 Free phosphate PO4 0.04 0.16 0.16 0.27 0.02 0.29 0.09 0.37

(84) The values in the table show that a phospholipase C L061 shows activity on PA, PC, PE and PI, at 60 C. and pH 5 and 6. Activity was also seen after 4 hours, after which hydrolysis had proceeded to 20-40% (values not reported).

Example 6. Hydrolysis of Phospholipids in Crude Soybean Oil at Neutral Conditions

(85) 100 gram crude soy bean oil (North American Expander, with about 2.16 wt % of total phospholipidsmeasured by NMR, see below) was brought to a temperature of 55 C. and first treated with 130 ppm NaOH (55 C. for 30 minutes), then 2.5% of water was added with enzyme obtained from shake flask fermentation (with 17.1 U/mL, see Example 3 above) at 3.14 L/g oil. This corresponds to 2.5 U PLC per gram phospholipid At time points 0.5, 2, 4 and 24 hr a sample was taken and the composition of phosphorous compounds was analyzed by .sup.31P NMR and diacylglyceride (DAG) levels by HPLC.

(86) .sup.31P NMR characterization was done in a slightly different protocol than as described in example 5.

(87) In the present example the TIP/TMP internal standard solution was prepared by mixing 100 mM Triisopropyl phosphate (TIP, 97%, Aldrich, #554669) and Trimethylphosphate (TMP, 99+%, Aldrich, #241024) solutions in 2-propanol (IPA, HPLC grade, J. T. Baker, #9095-33) respectively, at a ratio of 1:1.

(88) An extraction buffer was made with the same composition as the NMR solvent used for Example 5, except that the pH was brought to 10.5 using solid KOH. The extraction buffer was used to extract the phospholipids from the oil: An aliquot of 220 L of well stirred oil was weighed accurately into a 2 mL tube. 100 L D.sub.2O, and 900 L extracting buffer were added to the tube. The sample was mixed at room temperature and 1400 rpm for 60 minutes (benchtop Eppendorf Thermomixer R). After 60 minutes, the sample was centrifuged in a benchtop Eppendorf 5417C centrifuge at 14000 rpm for 10 minutes. 600 L of the aqueous extraction layer was transferred into a new tube, followed by accurate addition of 24 L of the TIP/TMP standard solution to the sample. The mixture was mixed using a benchtop vortex for 15 seconds. 500 L of this sample (accurately) was transferred into a clean NMR tube. NMR measurements were performed using a Bruker AVANCE 500 MHz NMR with QNP 500 MHz S2 5 mm probe with Z-gradient, using the .sup.31P Channel at 202.4 Hz and proton decoupling. The temperature was set at 300K. The number of scans was typically set at 512, and a relaxation delay at 0.1 s.

(89) Diacylglyceride levels were determined using a variant of the AOCS Official Method Cd 11d-96 by HPLC-ELSD, using an Agilent 1260 HPLC system with Agilent 380ELSD and Agilent Prep-SIL Scalar HPLC column (4.6150 mm, 10 m particle size; part#443910-901). Materials: Chemicals EMD Hexanes, part#HX0290P-1, EMD Ethyl Acetate, part#EX0245P-1, EMD Isopropyl Alcohol (IPA), part#PX1838P-1, EMD formic acid 98-100%, part#1116701000, Sigma Aldrich 1,2-Dipalmitoyl-rac-glycerol, 99%, part#D2135-1G, Sigma Aldrich Glyceryl 1,3-dipalmitate, 99%, part#D1639-1G.

(90) HPLC settings: Mobile phase A: Hexane/Mobile phase B: Hexane:Isopropanol:Ethyl Acetate:Formic Acid:800:100:100:1 and an elution gradient as follows:

(91) TABLE-US-00006 Time Mobile Phase A Mobile Phase B (min) (%) (%) 0.00 98.0 2.0 5.50 65.0 35.0 7.50 2.0 98.0 10.00 2.0 98.0

(92) Solvent Flow Rate: 2 mL/min, Operating Pressure: 30 bar; Column temperature: 40.0 C.; Injection Volume: 5.00 L

(93) Preparation of Oil Samples: An aliquot of 50 L oil sample was brought into 2 mL HPLC vial, accurately weighed and subsequently 950 L hexane/isopropanol (9:1) mixture was added to each oil sample. The sample was thoroughly mixed using a bench-top vortexer. This was measured against a standard from 1,2 DAG and 1,3 DAG in a 9:1 hexane:IPA mixture.

(94) The results in Tables 6 and 7 show that the polypeptide of the present disclosure had activity towards all phospholipids PA, PE, PI and PC present in crude soy bean oil under neutral conditions. Table 8 shows that the 1,2 diacylglyceride production ran parallel with the breakdown of intact phospholipids. These results as well as the decrease of intact phospholipid levels show that a polypeptide having phospholipase C activity according to the present invention is capable of increasing the oil yield in an enzymatic degumming process under neutral conditions.

(95) TABLE-US-00007 TABLE 6 Amount (in M) of phospholipids, phosphate and phosphate esters in soybean oil incubated with phospholipase L061 versus time. PO.sub.4 is the reaction product of the hydrolysis of PA. At t = 0 there is already some PO.sub.4 present in the starting oil Time (h) PA PE PI PC PO.sub.4 EP IP CP 0 866 1496 1202 2153 301 0 0 0 0.5 739 1572 1072 1979 447 35 147 394 2 723 1649 792 1495 739 143 494 1069 4 587 1425 602 973 903 250 533 1356 24 238 636 429 106 1604 832 600 1998

(96) TABLE-US-00008 TABLE 7 Amount of intact phospholipids in soybean oil incubated with phospholipase L061 versus time in mol % Time (h) PA PE PI PC 0 100 100 100 100 0.5 85.3 105.1 89.2 91.9 2 83.5 110.2 65.9 69.5 4 67.8 95.3 50.1 45.2 24 27.5 42.5 35.7 4.9

(97) TABLE-US-00009 TABLE 8 1,2-diacylglyceride and 1,3 diacylglyceride level in soybean oil incubated with phospholipase L061 in wt % versus time Time (h) 1,2 DAG 1,3 DAG 0 0.25 0.15 0.5 0.4 0.16 2 0.74 0.17 4 0.95 0.18 24 1.32 0.2

Example 7. Hydrolysis of Phospholipids in Crude Soybean Oil at Slightly Acidic Conditions

(98) A second reaction was run in the same way as described in the previous example, however now the oil was pretreated with 500 ppm citric and 138 ppm NaOH for acidic conditions and an enzyme concentration of 2.90 L/g oil, which corresponds to 2.3 U/g phospholipid was added.

(99) The results in Tables 9 and 10 show that the polypeptide having phospholipase according of the present disclosure had activity towards all phospholipids PA, PE, PI and PC present in crude soy bean oil under acid conditions. Table 11 shows that the 1,2 diacylglyceride production ran parallel with the breakdown of intact phospholipids.

(100) TABLE-US-00010 TABLE 9 Amount (in M) of phospholipids, phosphate and phosphate esters in soybean oil incubated with phospholipase L061 versus time Time (h) PA PE PI PC PO4 EP IP CP 0 866 1496 1202 2153 301 0 0 0 0.5 884 1692 1170 2216 427 0 138 224 2 705 1356 758 1286 523 62 332 548 4 671 1234 708 990 631 131 354 749 24 375 818 587 341 1111 363 436 1313

(101) TABLE-US-00011 TABLE 10 Amount (in mol %) of intact phospholipids in soybean oil incubated with phospholipase L061 versus time Time (h) PA PE PI PC 0 100 100 100 100 0.5 102.1 113.1 97.4 103.0 2 81.4 90.6 63.1 59.7 4 77.5 82.5 58.9 46.0 24 43.3 54.7 48.9 15.8

(102) TABLE-US-00012 TABLE 11 Diacylglyceride level (wt %) phospholipids in soybean oil incubated with phospholipase L061 versus time Time (h) 1,2 DAG 1,3 DAG 0 0.25 0.15 0.5 0.33 0.17 2 0.53 0.16 4 0.64 0.16 24 0.91 0.21

(103) The results in Example 6 and 7 show that a phospholipase of the present disclosure hydrolysed a higher amount of the phospholipids phosphatidyl choline, phosphatidyl ethanol amine, phosphatidyl inositol phosphatidic acid under neutral conditions than under acidic conditions.

Example 8. Hydrolysis of Phospholipids in Rape Seed Oil

(104) Extracted rape seed oil from European origin (100 mL) was pretreated with 200 ppm NaOH, and directly after that 5% w/w enzyme was added, that was obtained from a shake flask fermentation by treating broth with 10 mg Triton/gram broth, 60 min at room temperature and 10 minutes centrifugation at 4,000 rpm. The sample was sheared by a Silverson high shear mixer (1 minute at full speed) and left to incubate well stirred at 60 C. for 24 hours. During incubation samples were drawn.

(105) Samples were heat treated to inactivate the enzyme, frozen, freeze dried and treated with cold acetone to extract the polar lipids [10 mL acetone on 1 mL]. After drying the acetone precipitate was characterized by .sup.31NMR as described in example 5. The results are shown in table 12 (for clarity the lysophospholipids and glycerophosphates compounds are left out).

(106) TABLE-US-00013 TABLE 12 Phospholipid composition of extracted rapeseed oil incubated with phospholipase L061 versus time. Time free [hr] PC C-P PE E-P PI I-P PA PO.sub.4 mol/g 0 3.44 0.01 2.10 0.00 2.11 0.01 2.79 1.08 4 0.19 3.04 1.81 1.40 0.25 0.96 0.95 8.07 24 0.00 0.43 0.19 1.89 0.01 0.32 0.51 12.46 mol % 0 29.8 0.1 18.2 0.0 18.3 0.1 24.2 9.4 4 1.1 18.2 10.9 8.4 1.5 5.8 5.7 48.4 24 0.0 2.7 1.2 11.9 0.0 2.0 3.3 78.8

(107) The results in Table 12 show that the enzyme can hydrolyze all phospholipids in rape seed oil.

Example 9. Hydrolysis of Dairy Phospholipids

(108) Commercial dairy cream (Albert Heijn, Puur&Eerlijk) was freeze dried, washed with acetone to remove the non-polar lipids, redispersed in water and 5% enzyme solution obtained as described in Exampl 8 was added. After 4 hr at 50 C. the sample was freeze dried.

(109) This material was extracted with 3 ml CHCl.sub.3/MeOH on 100 mg dry sample weight. After taking out the extract/solvent the residue was washed with 2 ml CHCl.sub.3/MeOH, to yield the polar lipids and a part of the reaction products choline-phosphate, ethanolamine-phosphate, inositol phosphate and free phosphate (limited solubility in CHCl.sub.3/MeOH). These portions of CHCl.sub.3/MeOH were combined and dried under a stream of nitrogen. Subsequently the material was dissolved in the aqueous buffer and analyzed by .sup.31P NMR as described above.

(110) The results in Table 13 show that phospholipase C of the present disclosure hydrolyzes all phospholipids PC, PE, PI, PA and also phosphatidyl serine (PS).

(111) TABLE-US-00014 TABLE 13 Amount of phospholipids and reaction products in extracted dairy cream after treatment with phospholipase of the present disclosure PC C-P PS PE E-P PI I-P PA total In mol/g Ref 2.45 0.36 0.79 1.28 0.21 0.60 0.10 0.07 PLC 0.06 2.62 0.00 0.14 3.21 0.02 0.19 0.03 treated In mol % Ref 24.3 3.6 7.9 12.7 2.1 5.9 1.0 0.7 90.8 PLC 0.6 27.7 0.0 1.5 33.9 0.2 2.1 0.3 93.0 treated