Polypeptides having phospholipase C activity and polynucleotides encoding these polypeptides

Abstract

The present invention relates to methods for reducing the phospholipid content in oil or fat compositions and polypeptides having PI-specific phospholipase C activity capable of catalyzing such reduction, as well as polypeptides having PC, PE-specific phospholipase C activity and combinations thereof. The present invention also relates to polynucleotides encoding these polypeptides, nucleic acid constructs, vectors, and host cells comprising these polynucleotides, as well as methods for producing and using these polypeptides.

Description

[0001] References to sequence listings

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Background of the Invention

[0003] Field of the Invention

[0004] The present invention relates to methods for reducing the phospholipid content of oil compositions and polypeptides having phospholipase C activity that are capable of catalyzing such reduction. The present invention also relates to polynucleotides encoding these polypeptides, nucleic acid constructs, vectors, and host cells comprising these polynucleotides, as well as methods of producing and using these polypeptides.

[0005] Description of related fields

[0006] Several types of phospholipases are known, which differ in their specificity according to the position of the bond attacked in the phospholipid molecule. Phospholipase A1 (PLA1) removes the 1-position fatty acid to produce a free fatty acid and a 1-lyso-2-acylphospholipid. Phospholipase A2 (PLA2) removes the 2-position fatty acid to produce a free fatty acid and a 1-acyl-2-lysophospholipid. The term phospholipase B (PLB) is used for phospholipases that have both A1 and A2 activity. Phospholipase C (PLC) removes the phosphate moiety to produce 1,2-diacylglycerol and a phosphate ester. Phospholipase D (PLD) produces 1,2-diacylglycerol-phosphate and a base group (see Figure 1 ).

[0007] Before use, vegetable oil is degummed to provide refined storage stability vegetable oil, it has neutral taste and light color.Degumming process comprises removing phosphatide component (gum) from the oil fraction that is rich in triglyceride.The most commonly used method in industry is water degumming, chemical / caustic refining and physical refining that comprises acid-assisted degumming and / or enzyme-assisted degumming.Due to the emulsifying property of phosphatide component, degumming process causes the loss of oil, i.e. the loss of triglyceride.

[0008] Due to the effective hydrolysis of phospholipids that reduce emulsification, enzymatic degumming reduces oil loss. For a review of enzymatic degumming, see Dijkstra 2010, Eur. J. Lipid Sci. Technol. 112, 1178. The use of phospholipase A and / or phospholipase C in degumming is described, for example, in Clausen 2001, Eur. J. Lipid Sci. Technol. 103 333-340, WO 2003 / 089620 and WO 2008 / 094847. Phospholipase A solutions produce lysophospholipids and free fatty acids, resulting in oil loss. On the other hand, phospholipase C has the following advantages: it produces diglycerides ( Figure 2) and therefore will reduce losses. There are four major phospholipids in vegetable oils, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) and phosphatidylinositol (PI). Phospholipase C enzymes have different specificities for these phospholipids. The only known commercially available phospholipase C is Purifine (Dijkstra, the 101st AOCS Annual Meeting, May 10, 2010) from Van Enim / DSM, which has specificity for PC and PE. WO 07 / 059927 describes a thermotolerant Bacillus PLC for degumming. WO 2012 / 062817 describes a fungal PLC that is specific for all four phospholipids. PI-specific phospholipase C has been described in WO 2011 / 046815. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The locations where different phospholipases cleave phospholipids are illustrated along with the four main functional groups on phospholipids.

[0011] Figure 2 The reaction of phospholipids with phospholipase C to form diacylglycerol and phosphate or phosphoric acid is illustrated. Summary of the Invention

[0012] The present invention provides a method for reducing the phospholipid content in an oil composition, the method comprising

[0013] a) providing an oil composition comprising a certain amount of phospholipids,

[0014] b) contacting the oil composition with a phosphatidylinositol phospholipase C and a PC- and PE-specific phospholipase C under conditions sufficient for the enzymes to react with the phospholipids to produce a diacylglycerol and a phosphate ester, and

[0015] c) separating the phosphate ester from the oil composition. Specifically, the phosphatidylinositol phospholipase C is from Pseudomonas sp.

[0016] The present invention further provides a polypeptide having phosphatidylinositol phospholipase C activity, which is selected from the group consisting of:

[0017] a) a polypeptide having at least 91% sequence identity to the mature polypeptide of SEQ ID NO: 2;

[0018] b) a polypeptide encoded by a polynucleotide that hybridizes under moderate stringency conditions with

[0019] i) the mature polypeptide coding sequence of SEQ ID NO: 1, or

[0020] ii) the full-length complement of (i);

[0021] c) a polypeptide encoded by a polynucleotide having at least 90% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1;

[0022] d) a variant of the mature polypeptide of SEQ ID NO: 2, the variant comprising a substitution, deletion, and / or insertion at one or more positions; and

[0023] e) A fragment of the polypeptide of (a), (b), (c), or (d), which fragment has phosphatidylinositol phospholipase C activity.

[0024] In addition, the present invention provides a polypeptide having PC and PE specific phospholipase C activity, which is selected from the group consisting of:

[0025] a) a polypeptide having at least 70% sequence identity to the mature polypeptide of SEQ ID NO: 19;

[0026] b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions to

[0027] i) the mature polypeptide coding sequence of SEQ ID NO: 18, or

[0028] ii) the full-length complement of (i);

[0029] c) a polypeptide encoded by a polynucleotide having at least 70% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 18;

[0030] d) a variant of the mature polypeptide of SEQ ID NO: 19, the variant comprising a substitution, deletion, and / or insertion at one or more positions; and

[0031] e) A fragment of the polypeptide of (a), (b), (c), or (d), which fragment has PC- and PE-specific phospholipase C activity.

[0032] Finally, the present invention provides a composition comprising a phosphatidylinositol phospholipase C from Pseudomonas and a mixture of PC- and PE-specific phospholipase C polypeptides.

[0033] definition

[0034] Phospholipase C activity: The term "phospholipase C activity" or "PLC activity" refers to the activity of an enzyme that removes a phosphate moiety from a phospholipid to produce 1,2 diacylglycerol (see Figure 2). Most PLC enzymes belong to the hydrolase and phosphodiesterase families and are generally classified as EC 3.1.4.3. Some PLC enzymes are classified into other EC classes, for example, PI-specific PLC. Phospholipase C activity can be determined according to the procedure described in Example 5 or by one of the assays described in the "Phospholipase Activity Assay" section.

[0035] Phospholipase C specificity: The term "phospholipase C specificity" relates to a polypeptide having phospholipase C activity, wherein the activity is specific for one or more phospholipids, of which the four most important are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) and phosphatidylinositol (PI) (see Figure 1 ). Phospholipase C specificity can be determined by the method described in Example 5 32 confirmed by P-NMR.

[0036] PC and PE-specific phospholipase C: The term "PC and PE-specific phospholipase C" or "PC, PE-specific phospholipase C" relates to a polypeptide that has activity towards phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In addition to PC and PE-specificity, it may also have some activity towards phosphatidic acid (PA) and phosphatidylinositol (PI). Preferably, the PC and PE-specific phospholipase C removes at least 30% PC and at least 30% PE from an oil or fat having at least 100 ppm PC and 100 ppm PE when measured using the P-NMR assay of Example 5 at the enzyme's optimal pH and a dose of 10 mg / kg enzyme. More preferably, it removes 40%, 50%, 60%, 70% or 80%, even more preferably it removes 90%, and most preferably it removes between 90% and 100% of PC in oils and fats, and 40%, 50%, 60%, 70% or 80%, even more preferably it removes 90%, and most preferably it removes between 90% and 100% of PE in oils and fats.

[0037] PI-specific phospholipase C: The term "PI-specific phospholipase C" or "phosphatidylinositol phospholipase C" relates to a polypeptide that has activity on phosphatidylinositol (PI), meaning that its activity on phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) is low compared to its activity on PI. The PI-specific phospholipase C enzyme may belong to the family of hydrolases and phosphodiesterases classified as EC 3.1.4.11 or to the family of lyases classified as EC 4.6.1.13. The PI-specific phospholipase C activity may be determined according to the procedure described in Example 5. Preferably, the PI-specific phospholipase C removes at least 30% of the PI from an oil or fat having at least 50 ppm of PI when measured using the P-NMR of Example 5 at the enzyme's optimal pH and a dose of 10 mg / kg enzyme. More preferably, it removes 40%, 50%, 60%, 70% or 80%, even more preferably, it removes 90%, and most preferably it removes between 90% and 100% of the PI in the oil or fat.

[0038] Preferably, the PI-specific phospholipase C removes at least 20% more PI when compared to the amount of PC, PE or PA it can remove, more preferably at least 30%, 40%, even more preferably at least 50% and most preferably at least 60% more PI when compared to the amount of PC, PE or PA it can remove.

[0039] Allelic variant: The term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally from mutations and can lead to polymorphism within a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides with altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

[0040] Catalytic domain: The term "catalytic domain" means the region of an enzyme that contains the catalytic machinery of the enzyme.

[0041] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The first, primary RNA transcript is a precursor to mRNA that undergoes a series of processing steps, including splicing, before appearing as mature, spliced ​​mRNA.

[0042] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0043] Control sequences: The term "control sequences" means nucleic acid sequences necessary for the expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) to the polynucleotide encoding the polypeptide, or native or exogenous to each other. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters, and transcription and translation termination signals. For the purpose of introducing specific restriction enzyme sites that facilitate ligation of these control sequences to the coding region of the polynucleotide encoding the polypeptide, these control sequences may be provided with multiple linkers.

[0044] Crude oil: The term "crude oil" refers to (also known as non-degummed oil) pressed or extracted oils or mixtures thereof from, for example, vegetable sources, including but not limited to acai oil, almond oil, babassu oil, blackcurrent seed oil, borage seed oil, canola oil, cashew oil, castor oil, coconut oil, cilantro oil, corn oil, cottonseed oil, crambe oil, linseed oil, grapeseed oil, hazelnut oil, hempseed oil, jatropha oil, jojoba oil, linseed oil, macadamia nut oil, mango kernel oil, meadowfoam seed oil, mustard oil, neatsfoot oil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil, walnut oil, pine nut oil, pistachio oil, poppy seed oil, rapeseed oil, rice bran oil, safflower oil, camellia oil, sesame oil, avocado oil, soybean oil, sunflower oil, tall oil, tsubaki oil, oil), walnut oil, a variety of "natural" oils with altered fatty acid compositions from genetically modified organisms (GMOs) or traditionally "bred" such as high oleic, low linolenic or low saturate oils (high oleic canola, low linolenic soybean or high stearic sunflower).

[0045] Degummed oil: The term "degummed oil" refers to an oil obtained after removing non-hydratable phospholipids, hydratable phospholipids, and lecithin (collectively referred to as "gums") from the oil to produce a degummed oil or fat that can be used in food production and / or non-food applications, such as biodiesel. In certain embodiments, the degummed oil has a phospholipid content of less than 200 ppm phosphorus, such as less than 150 ppm phosphorus, less than 100 ppm phosphorus, less than (or less than about) 50 ppm phosphorus, less than (or less than about) 40 ppm phosphorus, less than (or less than about) 30 ppm phosphorus, less than (or less than about) 20 ppm phosphorus, less than (or less than about) 15 ppm phosphorus, less than (or less than about) 10 ppm phosphorus, less than (or less than about) 7 ppm phosphorus, less than (or less than about) 5 ppm phosphorus, less than (or less than about) 3 ppm phosphorus, or less than (or less than about) 1 ppm phosphorus.

[0046] Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0047] Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide operably linked to control sequences that provide for its expression.

[0048] Fragment: The term "fragment" means a polypeptide having one or more (e.g., several) amino acids missing from the amino and / or carboxyl terminus of a mature polypeptide or domain, wherein the fragment has phospholipase C activity. Fragments according to the present invention have a size of greater than about 200 amino acid residues, preferably greater than 210 amino acid residues, more preferably greater than 220 amino acid residues, more preferably greater than 230 amino acid residues (e.g., amino acids 44 to 278 or amino acids 34 to 268 of SEQ ID NO: 19), more preferably greater than 240 amino acid residues (e.g., amino acids 39 to 278 or amino acids 34 to 273 of SEQ ID NO: 19), more preferably greater than 250 amino acid residues, more preferably greater than 260 amino acid residues, more preferably greater than 270 amino acid residues, and most preferably greater than 280 amino acid residues. In one aspect, a fragment comprises at least 290 amino acid residues (e.g., amino acids 33 to 322 or amino acids 26 to 315 of SEQ ID NO: 2), at least 294 amino acid residues (e.g., amino acids 29 to 322 or amino acids 26 to 319 of SEQ ID NO: 2), or at least 296 amino acid residues (e.g., amino acids 27 to 322 or amino acids 26 to 321 of SEQ ID NO: 2).

[0049] High stringency conditions: The term "high stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 50% formamide. The support material is finally washed three times with 2X SSC, 0.2% SDS at 65°C for 15 minutes each.

[0050] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0051] Isolated: The term "isolated" means a material that is in a form or setting not found in nature. Non-limiting examples of isolated materials include (1) any non-naturally occurring material, (2) any material, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor, that is at least partially removed from one or more or all of the naturally occurring components with which it is essentially associated; (3) any material that has been artificially modified relative to the material found in nature; or (4) any material that has been modified by increasing the amount of the material relative to the other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the material; and use of a stronger promoter than the promoter naturally associated with the gene encoding the material).

[0052] Low stringency conditions: The term "low stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 25% formamide. The support material is finally washed three times with 2X SSC, 0.2% SDS at 50°C for 15 minutes each.

[0053] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and the like. In one aspect, based on the Signal P version 3 program (Nielsen et al., 1997, Protein Engineering 10:1-6), which predicts that amino acids 1 to 25 of SEQ ID NO: 2 are a signal peptide, the mature polypeptide is amino acids 26 to 322 of SEQ ID NO: 2. When expressed in Bacillus as described in Example 1, an additional alanine is added to the N-terminus. The N-terminal sequence of the mature polypeptide (SEQ ID NO: 3) was confirmed to be AQESPAF (see Example 3). On the other hand, based on the Signal P version 3 program (Nielsen et al., 1997, Protein Engineering 10:1-6) that predicts that amino acids 1 to 34 of SEQ ID NO: 19 are signal peptides, the mature polypeptide is amino acids 34 to 278 of SEQ ID NO: 19. When expressed in Bacillus as described in Example 1, additional alanine is added to the N-terminus. The N-terminal sequence of this mature polypeptide (SEQ ID NO: 20) is confirmed to be AWSADAP (see Example 3). As known in the art, a host cell can produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and therefore a host cell expressing a polynucleotide can produce different mature polypeptides (e.g., having different C-terminal and / or N-terminal amino acids) when compared to a host cell expressing the same polynucleotide. In one aspect, the mature polypeptide of SEQ ID NO: 2 comprises up to at least 300 amino acid residues (e.g., amino acids 23 to 322 of SEQ ID NO: 2), or at least 299 amino acid residues (e.g., amino acids 24 to 322 of SEQ ID NO: 2), or at least 298 amino acid residues (e.g., amino acids 25 to 322 of SEQ ID NO: 2), or at least 296 amino acid residues (e.g., amino acids 27 to 322 of SEQ ID NO: 2), or at least 295 amino acid residues (e.g., amino acids 28 to 322 of SEQ ID NO: 2).In another aspect, the mature polypeptide of SEQ ID NO: 19 comprises up to at least 247 amino acid residues (e.g., amino acids 31 to 278 of SEQ ID NO: 19), or at least 246 amino acid residues (e.g., amino acids 32 to 278 of SEQ ID NO: 19), or at least 244 amino acid residues (e.g., amino acids 35 to 278 of SEQ ID NO: 19), or at least 243 amino acid residues (e.g., amino acids 36 to 278 of SEQ ID NO: 19).

[0054] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide encoding a mature polypeptide having phospholipase activity. In one aspect, based on the prediction that nucleotides 1 to 75 of SEQ ID NO: 1 encode a signal peptide, SignalP (Nielsen et al., 1997, supra), the mature polypeptide coding sequence is nucleotides 76 to 966 of SEQ ID NO: 1. In another aspect, based on the prediction that nucleotides 1 to 99 of SEQ ID NO: 18 encode a signal peptide, SignalP (Nielsen et al., 1997, supra), the mature polypeptide coding sequence is nucleotides 100 to 837 of SEQ ID NO: 18.

[0055] Moderate stringency conditions: The term "moderate stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 35% formamide. The support material is finally washed three times with 2X SSC, 0.2% SDS at 55°C for 15 minutes each.

[0056] Medium to high stringency conditions: The term "medium to high stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 35% formamide. The support material is finally washed three times for 15 minutes each at 60°C using 2X SSC, 0.2% SDS.

[0057] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a manner not originally found in nature to contain a nucleic acid segment, or is synthesized, and includes one or more control sequences.

[0058] Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0059] Sequence identity: The parameter "sequence identity" is used to describe the relatedness between two amino acid sequences or between two nucleotide sequences.

[0060] For purposes of the present invention, use as in EMBOSS bag (EMBOSS: European Molecular Biology Open Software Suite (The European Molecular Biology Open Software Suite), Rice (Rice) et al., 2000, genetics trend (Trends Genet.) 16:276-277) (preferred 5.0.0 version or more recent version) Needleman-Wunsch (Needleman-Wunsch) algorithm (Needleman (Needleman) and Wunsch (Wunsch), 1970, Journal of Molecular Biology (J.Mol.Biol.) 48:443-453) determine the sequence identity between two amino acid sequences.The parameter used is gap opening penalty 10, gap extension penalty 0.5, and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.The output (using-non-simplified option is obtained) of " the longest consistency " of Needleman mark is used as percentage identity, and is calculated as follows:

[0061] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0062] For the purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wensch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, supra) (preferably version 5.0.0 or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of the "longest identity" labeled Needleman (obtained using the -non-simplified option) is used as percent identity and is calculated as follows:

[0063] (number of identical deoxyribonucleotides x 100) / (length of alignment - total number of gaps in the alignment)

[0064] Variant: The term "variant" refers to a polypeptide having phospholipase C activity that comprises an alteration (i.e., a substitution, insertion, and / or deletion at one or more (e.g., several) positions). Substitution means that the amino acid occupying a position is replaced with a different amino acid; deletion means removal of the amino acid occupying a position; and insertion means addition of an amino acid adjacent to and immediately following the amino acid occupying a position.

[0065] Very high stringency conditions: The term "very high stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 50% formamide. The support material is finally washed three times with 2X SSC, 0.2% SDS at 70°C for 15 minutes each.

[0066] Nucleic acid sequence and amino acid sequence

[0067] SEQ ID NO: 1: PI-specific PLC, Pseudomonas sp.; coding sequence.

[0068] SEQ ID NO: 2: PI-specific PLC, Pseudomonas sp.; amino acid sequence

[0069] SEQ ID NO: 3: PI-specific PLC, Pseudomonas sp.; amino acid sequence of the mature polypeptide with an N-terminal alanine.

[0070] SEQ ID NO: 4: PI-specific PLC, Pseudomonas chlorapsis; coding sequence.

[0071] SEQ ID NO: 5: PI-specific PLC, Pseudomonas chlororaphis; amino acid sequence.

[0072] SEQ ID NO: 6: PI-specific PLC, Pseudomonas chlororaphis; amino acid sequence of the mature polypeptide with an N-terminal alanine.

[0073] SEQ ID NO: 7: PI-specific PLC, Pseudomonas sp.; coding sequence.

[0074] SEQ ID NO: 8: PI-specific PLC, Pseudomonas sp.; amino acid sequence.

[0075] SEQ ID NO: 9: PI-specific PLC, Pseudomonas sp.; amino acid sequence of the mature polypeptide with an N-terminal alanine.

[0076] SEQ ID NO: 10: PI-specific PLC, Pseudomonas chlororaphis; coding sequence.

[0077] SEQ ID NO: 11: PI-specific PLC, Pseudomonas chlororaphis; amino acid sequence.

[0078] SEQ ID NO: 12: PI-specific PLC, Pseudomonas chlororaphis; amino acid sequence of the mature polypeptide with an N-terminal alanine.

[0079] SEQ ID NO: 13: PI-specific PLC, Pseudomonas protegens; coding sequence.

[0080] SEQ ID NO: 14: PI-specific PLC, Pseudomonas proteoglycans; amino acid sequence.

[0081] SEQ ID NO: 15: PI-specific PLC, Pseudomonas proteoglycans; amino acid sequence of the mature polypeptide with an N-terminal alanine.

[0082] SEQ ID NO: 16: PI-specific PLC, Pseudomonas proteoglycans; coding sequence.

[0083] SEQ ID NO: 17: PI-specific PLC, Pseudomonas proteoglycans; amino acid sequence.

[0084] SEQ ID NO: 18: PC / PE-specific PLC, Bacillus sp. encoding sequence.

[0085] SEQ ID NO: 19: PC / PE-specific PLC, Bacillus sp. amino acid sequence.

[0086] SEQ ID NO: 20: Amino acid sequence of PC / PE-specific PLC, Bacillus sp. mature polypeptide with N-terminal alanine.

[0087] SEQ ID NO: 21: Bt-PLC Bacillus thuringiensis; coding sequence.

[0088] SEQ ID NO: 22: Bacillus thuringiensis; amino acid sequence of mature polypeptide

[0089] SEQ ID NO: 23: PC / PE-specific PLC, Bacillus pseudomycoides; coding sequence.

[0090] SEQ ID NO: 24: PC / PE-specific PLC, Bacillus pseudomycoides; amino acid sequence.

[0091] SEQ ID NO: 25: PC / PE-specific PLC, Bacillus pseudomycoides; amino acid sequence with N-terminal alanine.

[0092] SEQ ID NO: 26: PC / PE-specific PLC, Bacillus mycoides; coding sequence.

[0093] SEQ ID NO: 27: PC / PE-specific PLC, Bacillus mycoides; amino acid sequence.

[0094] SEQ ID NO: 28: PC / PE-specific PLC, Listeria innocua; coding sequence.

[0095] SEQ ID NO: 29: PC / PE-specific PLC, Listeria innocua; amino acid sequence.

[0096] SEQ ID NO: 30: PC / PE-specific PLC, Listeria innocua; amino acid sequence with N-terminal alanine.

[0097] SEQ ID NO:31: SEQ ID NO:3 of WO 2011 / 046812.

[0098] SEQ ID NO:32: SEQ ID NO:4 of WO 2011 / 046812.

[0099] SEQ ID NO: 33: Heterologous signal peptide

[0100] SEQ ID NO:34: PI-specific PLC, Amycolatopsis azurea; coding sequence

[0101] SEQ ID NO: 35: PI-specific PLC, Amycolatopsis truncatula; amino acid sequence

[0102] SEQ ID NO: 36: PI-specific PLC, Amycolatopsis farcifolius; amino acid sequence with N-terminal alanine.

[0103] SEQ ID NO: 37: PC / PE-specific PLC, Bacillus macauensis; coding sequence

[0104] SEQ ID NO: 38: PC / PE-specific PLC, Bacillus macau; amino acid sequence.

[0105] Detailed description of the invention

[0106] The present invention relates to phospholipase C enzymes obtained from various strains belonging to the genus Pseudomonas, which are non-pathogenic (class 1) organisms and are therefore generally considered safe for use in the laboratory. The phospholipase C enzymes derived from Pseudomonas strains all show specificity for PI.

[0107] The present invention also relates to PC, PE-specific phospholipase C enzymes that are novel or have never been expressed or characterized. The use of the PC, PE-specific phospholipase C, Purifine PLC, in degumming is known, however, it is still relevant to identify additional PC, PE-specific phospholipase C enzymes that perform well in degumming. In preferred embodiments, the PC, PE-specific phospholipase is obtained from a strain belonging to the genus Bacillus or Listeria.

[0108] The present invention further relates to methods for reducing the phospholipid content in oil compositions using one or more bacterial phospholipase C enzymes. Specifically, when PI-specific phospholipase C enzymes are combined with PC, PE-specific phospholipase C enzymes, beneficial effects related to the removal of phospholipids from oil compositions are observed.

[0109] Polypeptide with phospholipase C activity

[0110] One aspect of the present invention relates to a polypeptide having phosphatidylinositol phospholipase C activity, which polypeptide is selected from the group consisting of: a) a polypeptide having at least 91% sequence identity to the mature polypeptide of SEQ ID NO: 2; b) a polypeptide encoded by a polynucleotide that hybridizes under moderate stringency conditions with i) the mature polypeptide coding sequence of SEQ ID NO: 1, or ii) the full-length complement of (i); c) a polypeptide encoded by a polynucleotide having at least 90% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1; d) a variant of the mature polypeptide of SEQ ID NO: 2, the variant comprising a substitution, deletion, and / or insertion at one or more positions; and e) a fragment of the polypeptide of (a), (b), (c), or (d), which fragment has phosphatidylinositol phospholipase C activity.

[0111] In one embodiment, the present invention relates to polypeptides having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 2, which polypeptide is specific for phosphatidylinositol. In one aspect, these polypeptides differ from the mature polypeptide of SEQ ID NO: 2 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids. In a preferred embodiment, the mature polypeptide of SEQ ID NO: 2 corresponds to amino acids 26 to 322 of SEQ ID NO: 2.

[0112] In one embodiment, the polypeptide has been isolated. The polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO:2 or an allelic variant thereof, or is a fragment thereof having phosphatidylinositol phospholipase C activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO:2. In another aspect, the polypeptide comprises or consists of amino acids 26 to 322 of SEQ ID NO:2 or amino acids 1 to 298 of SEQ ID NO:3.

[0113] In particular, the polypeptide may have a length of 280-320 amino acid residues, such as 280-310 amino acid residues, 280-305 amino acid residues, 280-300 amino acid residues, 280-298 amino acid residues, 280-297 amino acid residues, 280-296 amino acid residues, 285-320 amino acid residues, 285-315 amino acid residues, 285-310 amino acid residues, 285-305 amino acid residues, 285-300 amino acid residues, 285-298 amino acid residues, 285-297 amino acid residues, 285-296 amino acid residues, 29 or a length of 0-320 amino acid residues, 290-315 amino acid residues, 290-310 amino acid residues, 290-305 amino acid residues, 290-300 amino acid residues, 290-298 amino acid residues, 290-297 amino acid residues, 290-296 amino acid residues, 295-320 amino acid residues, 295-315 amino acid residues, 295-310 amino acid residues, 295-305 amino acid residues, 295-300 amino acid residues, 295-298 amino acid residues, 255-297 amino acid residues, or 295-296 amino acid residues in length.

[0114] In a preferred embodiment, when used in 10 mg enzyme protein / kg oil at the optimal pH of the polypeptide, the phosphatidylinositol phospholipase C of the present invention can reduce the PI content in the crude oil by at least 30%, more preferably at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. In another embodiment, the optimal pH range of the polypeptide of the present invention is between 4.5 and 8.5, more preferably from 5.0 to 8.0, even more preferably from 6.5 to 7.5.

[0115] According to some embodiments, the phosphatidylinositol phospholipase C of the invention has a thermal denaturation temperature of at least 60°C, such as 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C or at least 90°C as determined by differential scanning calorimetry (DSC).

[0116] The denaturation temperature can be specifically determined as the apex of the denaturation peak (major endothermic peak) in the thermogram (Cp vs. T) obtained after heating a 1 mg / ml solution of the polypeptide in buffer (50 mM Na-acetoacetate (pH 5.5), or 50 mM Hepes (pH 7)) at a constant programmed heating rate of 200 K / hr.

[0117] According to some embodiments, the phosphatidylinositol phospholipase C of the invention is capable of reducing the phosphatidylinositol content of crude soybean oil by 50% or more, such as 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more, the reduction in phosphatidylinositol content being achieved after adding 100 mg enzyme protein (EP) / kg oil and incubating the oil and enzyme at 50° C. at pH 5.5 for 2 hours. 31 Confirmed by P-NMR.

[0118] Preferably, the phosphatidylinositol phospholipase C of the present invention is capable of reducing the phosphorus content of crude soybean oil to 20 mg / kg oil or less as measured by inductively coupled plasma optical emission spectroscopy (ICP-OES) after incubation of 4 mg enzyme protein / kg oil in a low-aqueous system comprising 3% water based on the amount of oil at 50°C-60°C for 5 hours.

[0119] For the purpose of determining the ability of the polypeptide to reduce phosphorus, crude soybean oil can be used, which comprises 80-140 ppm of phosphorus present as phosphatidic acid (PA), 140-200 ppm of phosphorus present as phosphatidylethanolamine (PE), 70-110 ppm of phosphorus present as phosphatidic acid (PI) and 130-200 ppm of phosphorus present as phosphatidylcholine; the phosphorus content is determined by 31 P-NMR measurement.

[0120] In further embodiments, the phosphatidylinositol phospholipase C of the invention is stable to different pH conditions and shows good performance in water degumming (non-acid / alkaline) as well as acid-assisted degumming followed by alkaline neutralization with different concentrations of NaOH (e.g., by adding orthophosphoric acid in an amount equivalent to 0.05% (100% pure orthophosphoric acid) based on the amount of oil, followed by alkaline neutralization with 0.5 equivalents, 1.0 equivalents or 1.5 equivalents of NaOH).

[0121] Furthermore, a reduction in phosphorus content can be obtained in an oil degumming process comprising the steps of:

[0122] i) optionally treating crude soybean oil with acid / base by adding 85% orthophosphoric acid solution in an amount corresponding to 0.05% (100% pure orthophosphoric acid) based on the amount of oil, mixing in an ultrasonic bath for 5 minutes, followed by incubation in a rotator for 15 minutes and neutralization with 4M NaOH base in an amount equivalent to pure orthophosphoric acid (from 0.5 to 0.15) in an ultrasonic bath for 5 minutes;

[0123] ii) adding the polypeptide to the oil in an amount of 4 mg enzyme protein / kg oil in a low-water system including 3% water based on the amount of oil, and subjecting the oil and the polypeptide to ultrasonic treatment for 5 minutes;

[0124] iii) incubating the polypeptide and oil at 50° C.-60° C. for 5 hours with stirring at 20 rpm;

[0125] iv) Centrifuge the oil and the polypeptide at 700 g at 85°C for 15 minutes.

[0126] Homologs of the PI-specific phospholipase C of the present invention have been identified from sequences annotated in genome sequencing projects. When aligned with the mature sequence of SEQ ID NO: 2, the identities are as follows:

[0127]

[0128] To the best of our knowledge, none of these homologs have been expressed or characterized, and their use in degumming or any other application has never been described. For the purposes of generating nucleic acid constructs, expression vectors, and host cells, as well as compositions and methods of use, the PI-specific phospholipase C of SEQ ID NO: 2, together with homologs of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 17, are PI-specific phospholipase C polypeptides of the invention, and the polynucleotides encoding them are polynucleotides of the invention.

[0129] In another embodiment, the present invention relates to a polypeptide having PI-specific phospholipase C activity, which polypeptide is encoded by a polynucleotide that hybridizes to (i) the mature polypeptide coding sequence of SEQ ID NO: 1, or (ii) the full complement of (i) or (ii) under medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor, New York). In one embodiment, the polypeptide has been isolated.

[0130] The polynucleotides of SEQ ID NO: 1, 4, 7, 10, 13 or 16 or subsequences thereof, together with the polypeptides of SEQ ID NO: 2, 5, 8, 11, 14 or 17 or fragments thereof, can be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having PI-specific phospholipase C activity from strains of different genera or species according to methods well known in the art. Specifically, such probes can be hybridized with genomic DNA or cDNA of cells of interest following standard Southern blotting procedures to identify and isolate the corresponding genes therein. Such probes can be significantly shorter than the complete sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probe is typically labeled (e.g., with a 32 P. 3 H. 35 S, biotin, or avidin) to detect the corresponding gene. The present invention covers such probes.

[0131] Can be directed to the DNA of the polypeptide with PI-specific phospholipase C activity that hybridizes with the probe described above and encodes, genomic DNA or cDNA library prepared from other bacterial strains of this type are screened. Genomic DNA or other DNA from other bacterial strains of this type can be separated by agarose or polyacrylamide gel electrophoresis or other separation techniques. DNA from the library or isolated DNA can be transferred to and be fixed on nitrocellulose or other suitable carrier materials. In order to identify clones or DNA that hybridize with SEQ ID NO:1 or its subsequences, carrier materials are used for southern blotting.

[0132] For the purposes of the present invention, hybridization means hybridization of a polynucleotide to a labeled nucleic acid probe corresponding to: (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1; (iii) its full-length complement; or (iv) a subsequence thereof; the hybridization being conducted under very high stringency conditions. Molecules that hybridize to the nucleic acid probe under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.

[0133] In another embodiment, the present invention relates to a polypeptide having PI-specific phospholipase C activity, which is encoded by a polynucleotide having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1. In further embodiments, the polypeptide has been isolated.

[0134] In another embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO:2 comprising substitutions, deletions, and / or insertions at one or more (e.g., several) positions. A specific variant of SEQ ID NO:2 is disclosed as SEQ ID NO:3 comprising an insertion of A before Q in position 26 of SEQ ID NO:2. In one embodiment, the number of amino acid substitutions, deletions, and / or insertions introduced into the mature polypeptide of SEQ ID NO:2 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. These amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions of typically 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by changing the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding domain.

[0135] Another aspect of the invention relates to a polypeptide having PC and PE-specific phospholipase C activity, which polypeptide is selected from the following group, which consists of: a) a polypeptide having at least 70% sequence identity with the mature polypeptide of SEQ ID NO: 19; b) a polypeptide encoded by the following polynucleotide, which hybridizes under low stringency conditions with i) the mature polypeptide coding sequence of SEQ ID NO: 18, or ii) the full-length complement of (i); c) a polypeptide encoded by a polynucleotide, which has at least 70% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 18; d) a variant of the mature polypeptide of SEQ ID NO: 19, which variant comprises a substitution, deletion, and / or insertion at one or more positions; and e) a fragment of the polypeptide of (a), (b), (c), or (d), which fragment has PC and PE-specific phospholipase C activity.

[0136] In one embodiment, the invention relates to polypeptides having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 19, which polypeptides have PC and PE-specific phospholipase C activity. In one aspect, these polypeptides differ from the mature polypeptide of SEQ ID NO: 19 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids.

[0137] In embodiments, the polypeptide has been isolated. The polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO: 19 or an allelic variant thereof, or a fragment thereof having phospholipase C activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 19. In another aspect, the polypeptide comprises or consists of amino acids 34 to 278 of SEQ ID NO: 19 or amino acids 1 to 246 of SEQ ID NO: 20.

[0138] In further embodiments, the polypeptide has a length of 220-280 amino acid residues, such as 220-270 amino acid residues, 220-260 amino acid residues, 220-250 amino acid residues, 220-248 amino acid residues, 220-246 amino acid residues, 220-244 amino acid residues, 225-280 amino acid residues, 225-270 amino acid residues, 225-260 amino acid residues, 225-250 amino acid residues, 225- 248 amino acid residues, 225-246 amino acid residues, 225-244 amino acid residues, 230-280 amino acid residues, 230-270 amino acid residues, 230-260 amino acid residues, 230-250 amino acid residues, 230-248 amino acid residues, 230-246 amino acid residues, 230-244 amino acid residues, 235-280 amino acid residues, 235-270 amino acid residues, 235-260 amino acid residues , 235-250 amino acid residues, 235-248 amino acid residues, 235-246 amino acid residues, 235-244 amino acid residues, 240-280 amino acid residues, 240-270 amino acid residues, 240-260 amino acid residues, 240-250 amino acid residues, 240-248 amino acid residues, 240-246 amino acid residues, 240-244 amino acid residues, 242-280 amino acid residues, 242-270 amino acid residues, 242-260 amino acid residues, 242-250 amino acid residues, 242-248 amino acid residues, 242-246 amino acid residues, 242-244 amino acid residues, 243-280 amino acid residues, 243-270 amino acid residues, 243-260 amino acid residues, 243-250 amino acid residues, 243-248 amino acid residues, 243-246 amino acid residues, 243-244 amino acid residues in length.

[0139] In particular, the polypeptide having PC and PE-specific phospholipase C activity can have a thermal denaturation temperature of at least 60°C, such as 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C or at least 90°C as determined by differential scanning calorimetry (DSC).

[0140] The denaturation temperature can be determined as the apex of the denaturation peak (major endothermic peak) in the thermogram (Cp vs. T) obtained after heating a 1 mg / ml solution of the polypeptide in buffer (50 mM Na-acetoacetate (pH 5.5), or 50 mM Hepes (pH 7)) at a constant programmed heating rate of 200 K / hr.

[0141] In a preferred embodiment, the PC and PE-specific phospholipase C of the present invention can reduce the PC and PE content in crude oil. Preferably, when used at 10 mg enzyme protein / kg oil at the optimal pH of the polypeptide, the polypeptide of the present invention can reduce the PC and PE content in the crude oil by at least 30%, more preferably at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. In another embodiment, the optimal pH range of the polypeptide of the present invention is between 5.0 and 8.5, more preferably from 5.5 to 8.0, even more preferably from 6.0 to 7.5. In further embodiments, the polypeptide having PC and PE-specific phospholipase C activity is capable of reducing the phosphatidylethanolamine and / or phosphatidylcholine content of crude soybean oil by 50% or more, such as 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more, the reduction in phosphatidylethanolamine and / or phosphatidylcholine content being achieved after adding 100 mg enzyme protein (EP) / kg oil and incubating the oil and enzyme at 50° C. at pH 5.5 for 2 hours. 31 Confirmed by P-NMR.

[0142] In yet further embodiments, the polypeptide having PC- and PE-specific phospholipase C activity is capable of reducing the phosphorus content of crude soybean oil to 20 mg / kg oil or less as measured by inductively coupled plasma optical emission spectroscopy (ICP-OES) after incubation of 4 mg enzyme protein / kg oil at 50°C-60°C for 5 hours in a low aqueous system comprising 3% water based on the amount of oil.

[0143] Specifically, the ability of the polypeptide to reduce phosphorus content can be determined using crude soybean oil comprising 80-140 ppm phosphorus present as phosphatidic acid (PA), 140-200 ppm phosphorus present as phosphatidylethanolamine (PE), 70-110 ppm phosphorus present as phosphatidic acid (PI), and 130-200 ppm phosphorus present as phosphatidylcholine; phosphorus content is determined by 31 P-NMR measurement.

[0144] In particular, a reduction in phosphorus content and / or a reduction in phosphatidylethanolamine and / or phosphatidylcholine content can be obtained in an oil degumming process comprising the following steps:

[0145] i) optionally treating crude soybean oil with acid / base by adding 85% orthophosphoric acid solution in an amount corresponding to 0.05% (100% pure orthophosphoric acid) based on the amount of oil, mixing in an ultrasonic bath for 5 minutes, followed by incubation in a rotator for 15 minutes and neutralization with 4M NaOH base in an amount equivalent to pure orthophosphoric acid (from 0.5 to 0.15) in an ultrasonic bath for 5 minutes;

[0146] ii) adding the polypeptide to the oil in an amount of 4 mg enzyme protein / kg oil in a low-water system including 3% water based on the amount of oil, and subjecting the oil and the polypeptide to ultrasonic treatment for 5 minutes;

[0147] iii) incubating the polypeptide and oil at 50° C.-60° C. for 5 hours with stirring at 20 rpm;

[0148] iv) Centrifuge the oil and the polypeptide at 700 g at 85°C for 15 minutes.

[0149] The closest related sequence to the PC, PE-specific PLC of the present invention is SEQ ID NO: 4 from WO 2011 / 046812 (SEQ ID NO: 32 in the present application), which has 44.6% identity to the mature sequence of SEQ ID NO: 19 (amino acids 34 to 278). This, along with the PC, PE-specific PLC identified below by UniProt numbering, can be used in the methods of the present invention.

[0150]

[0151] With respect to degumming, the mature sequence of SEQ ID NO:22 corresponding to amino acids 39 to 283 has also been disclosed as SEQ ID NO:5 in EP1788080. To the best of our knowledge, none of the PC, PE-specific PLCs of SEQ ID NO:24, 27 or 29 have been expressed or characterized, and their use in degumming or any other application has never been described. For the purposes of generating nucleic acid constructs, expression vectors, and host cells, as well as compositions and methods of use, the PC, PE-specific phospholipase C of SEQ ID NO:19, together with homologs of SEQ ID NO:24, SEQ ID NO:27 and SEQ ID NO:29, are PC, PE-specific phospholipase C polypeptides of the present invention, and the polynucleotides encoding them are polynucleotides of the present invention.

[0152] In another embodiment, the present invention relates to a polypeptide having PC and PE-specific phospholipase C activity, which polypeptide is encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 18, or (ii) the full-length complement of (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor, New York). In one embodiment, the polypeptide has been isolated.

[0153] The polynucleotide of SEQ ID NO: 18 or a subsequence thereof, together with the polypeptide of SEQ ID NO: 19 or a fragment thereof can be used to design nucleic acid probes to identify and clone DNA encoding a polypeptide having phospholipase C activity from strains of different genera or species according to methods well known in the art. Specifically, such probes can be hybridized with the genomic DNA or cDNA of cells of interest using standard Southern blotting procedures to identify and isolate the corresponding gene therein. Such probes can be significantly shorter than the complete sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probe is typically labeled (e.g., with a 32 P. 3 H. 35 S, biotin, or avidin) to detect the corresponding gene. The present invention covers such probes.

[0154] Can be directed against the DNA of the polypeptide with PC and PE-specific phospholipase C activity with above-described probe hybridization and encoding, genomic DNA or cDNA library prepared from this type of other bacterial strains are screened. Genomic DNA or other DNA from this type of other bacterial strains can be separated by agarose or polyacrylamide gel electrophoresis or other separation techniques. DNA from the library or isolated DNA can be transferred to and be fixed on nitrocellulose or other suitable carrier materials. In order to identify clones or DNA hybridized with SEQ ID NO:18 or its subsequences, carrier materials are used for southern blotting.

[0155] For the purposes of the present invention, hybridization means that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 18; (ii) the mature polypeptide coding sequence of SEQ ID NO: 18; (iii) its full-length complement; or (iv) a subsequence thereof under low to very high stringency conditions. Molecules that hybridize to the nucleic acid probe under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.

[0156] In another embodiment, the present invention relates to a polypeptide having PC and PE-specific phospholipase C activity, encoded by a polynucleotide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 18. In further embodiments, the polypeptide has been isolated.

[0157] In another embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO: 19 comprising substitutions, deletions, and / or insertions at one or more (e.g., several) positions. A specific variant of SEQ ID NO: 19 is disclosed as SEQ ID NO: 20 comprising an insertion of A before W in position 34 of SEQ ID NO: 19. In one embodiment, the number of amino acid substitutions, deletions, and / or insertions introduced into the mature polypeptide of SEQ ID NO: 19 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. These amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions of typically 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, e.g., an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by altering the net charge or another function, e.g., a polyhistidine stretch, an antigenic epitope, or a binding domain.

[0158] With respect to the polypeptides of the present invention, examples of conservative substitutions are within the following groups: the basic amino acid group (arginine, lysine, and histidine), the acidic amino acid group (glutamic acid and aspartic acid), the polar amino acid group (glutamine and asparagine), the hydrophobic amino acid group (leucine, isoleucine, and valine), the aromatic amino acid group (phenylalanine, tryptophan, and threonine), and the small amino acid group (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0159] Other possible approaches to generate variants having similar or substantially similar physicochemical and functional properties to the PI-specific phospholipase C of SEQ ID NO: 2 (mature polypeptide of SEQ ID NO: 2) or the PC PE-specific phospholipase C of SEQ ID NO: 19 (mature polypeptide of SEQ ID NO: 19) would include introducing changes into the amino acid sequence in regions showing moderate to high variability, identified by aligning the respective polypeptides with related sequences. In the PI-specific phospholipase C of SEQ ID NO: 2, such regions can be identified by best fit alignment with the amino acid sequences of SEQ ID NOs: 8 (UniProtKB / TrEMBL: J3EBR2), 11 (UniProtKB / TrEMBL: I4Y4N5), 14 (UniProtKB / TrEMBL: Q4K3U9), and 17 (UniProtKB / TrEMBL: R4RTF9).

[0160] Through this alignment, the following regions of medium or high variability can be identified in SEQ ID NO: 2 (using the amino acid code of SEQ ID NO: 2):

[0161]

[0162]

[0163] Thus, in some embodiments of the invention, a polypeptide has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 2, which is specific for phosphatidylinositol, wherein, encoded by the amino acids in SEQ ID NO: 2, one or more amino acid residues have been substituted, deleted, or added in one or more regions defined by amino acids 28-43, amino acids 59, amino acids 82-88, amino acids 130-131, amino acids 266, amino acids 298-301, amino acids 311-314.

[0164] In further embodiments, these polypeptides differ from the mature polypeptide of SEQ ID NO:2 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids.

[0165] In the corresponding embodiment for the PE / PC-specific phospholipase C of SEQ ID NO: 19, such regions can be identified by best fit alignment with the amino acid sequences of SEQ ID NOs: 22 (UniProtKB / TrEMBL: C3HDV6), 24 (UniProtKB / TrEMBL: C3BG10), 27 (UniProtKB / TrEMBL: C3AHL7) and 29 (UniProtKB / TrEMBL: E3Z3X0).

[0166] From this alignment, the following regions of medium or high variability can be identified in SEQ ID NO: 19 (using the amino acid code of SEQ ID NO: 19):

[0167]

[0168]

[0169] In one embodiment, the present invention relates to polypeptides having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 19, which have PC and PE-specific phospholipase C activity, wherein the polypeptide is expressed using SEQ ID NO: 19. The amino acids in NO:19 encode one or more amino acid residues that have been substituted, deleted or added, such as up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, in one or more regions defined by amino acids 3-72, amino acids 74-123, amino acids 129-142, amino acids 145-147, amino acids 154-155, amino acids 164-189, amino acids 188-201, amino acids 203-226, amino acids 228-237, amino acids 244-246, amino acids 248-258, amino acids 260-278.

[0170] In further embodiments, these polypeptides differ from the mature polypeptide of SEQ ID NO: 19 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids.

[0171] Alternatively, the amino acid change has such a property that the physicochemical properties of the polypeptide are altered. For example, the amino acid change can improve the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, etc.

[0172] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are tested for the desired phospholipase C activity to identify amino acid residues that are critical for the activity of the molecule. See also Hilton et al., 1996, J. Biol. Chem., 271: 4699-4708. Mutations in putative contact site amino acids can also be combined with physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, to determine the active site of the enzyme or other biological interactions. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of essential amino acids can also be inferred from alignments with related polypeptides.

[0173] The essential amino acid predictions in the sequence of amino acid 26 to 322 of SEQ ID NO:2 are located at positions H50, N51, and D74. It is believed that these amino acids are related to the calcium that coordinates the active site. This prediction is supported by the following article, Iwasaki et al., 1998, Biochimica et Biophysica Acta 1391:52-66, which demonstrates that when the H corresponding to the H50 in SEQ ID NO:2 is changed, the PLC identified as UniProt B3A043 or PDB 3H4X disappears. In a preferred embodiment, when compared with SEQ ID NO:2, polypeptide of the present invention maintains the amino acid corresponding to positions 50, 51, and 74.

[0174] Essential amino acids in the sequence of amino acids 34 to 278 of SEQ ID NO: 19 are predicted to be located at positions W34, H47, D88, H102, H152, D156, H162, H177, and E181. Based on a homology model of the sequence, it is believed that these amino acids are involved in coordinating the three Zn ions required for catalytic activity. In a preferred embodiment, when aligned with SEQ ID NO: 19, the polypeptide of the present invention retains the amino acids corresponding to positions 34, 47, 88, 102, 152, 156, 162, 177, and 181.

[0175] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested using known methods of mutagenesis, recombination and / or shuffling, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0176] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0177] The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused to the N-terminus or C-terminus of a region of another polypeptide.

[0178] Polypeptide can also be fusion polypeptide or cleavable fusion polypeptide, wherein another polypeptide is fused to the N-terminal or C-terminal of the polypeptide of the present invention.By fusing the polynucleotide encoding another polypeptide to the polynucleotide of the present invention, fusion polypeptide is produced.Technology for producing fusion polypeptide is known in the art, and includes connecting the coding sequence of the coded polypeptide so that they are in frame and the expression of the fusion polypeptide is under the control of the same one or more promoters and terminators.Fusion polypeptide can also be constructed using intein technology, wherein the fusion polypeptide is produced after translation (Cooper et al., 1993, European Molecular Biology Association Journal (EMBO J.) 12:2575-2583; Dawson et al., 1994, Science (Science) 266:776-779).

[0179] The fusion polypeptide may further include a cleavage site between the two polypeptides. When the fusion protein is secreted, the site is cleaved, thereby releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0180] Sources of polypeptides having phospholipase C activity

[0181] The polypeptides having phospholipase C activity of the present invention can be obtained from microorganisms of any genus. For the purposes of the present invention, the term "obtained from" as used herein in conjunction with a given source shall mean that the polypeptide encoded by the polynucleotide is produced by the source or by a strain into which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0182] The polypeptide can be a bacterial polypeptide. For example, the polypeptide can be a Gram-positive bacterial polypeptide, such as a Bacillus, Listeria or Pseudomonas polypeptide.

[0183] In another aspect, the polypeptide is an alkalophilic Bacillus, amyloliquefaciens, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus laurens, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pseudomycoides, Bacillus brevis, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide.

[0184] In another aspect, the polypeptide is a Listeria innocua polypeptide.

[0185] In another aspect, the polypeptide is a Pseudomonas chlororaphis or Pseudomonas proteoglycans polypeptide.

[0186] It will be understood that for the aforementioned species, the present invention encompasses perfect and imperfect states, and other taxonomic equivalents, such as anamorphs, regardless of the species name by which they are known. One of ordinary skill in the art will readily recognize the identity of appropriate equivalents.

[0187] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), the Netherlands Center for the Study of Microorganisms (Centraalbureau Voor Schimmelcultures, CBS), and the U.S. Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0188] The above-mentioned probe can be used from other sources, including microorganisms isolated from nature (for example, soil, compost, water, etc.) or directly from natural materials (for example, soil, compost, water, etc.) to identify and obtain the polypeptide. The technology for directly isolating microorganisms and DNA from natural living environments is well known in the art. The polynucleotide encoding the polypeptide can then be obtained by similarly screening the genomic DNA or cDNA library of another microorganism or the DNA sample of the mixture. Once the polynucleotide encoding the polypeptide is detected with one or more probes, the polynucleotide can be separated or cloned by using technology known to those of ordinary skill in the art (see, for example, Sambrook et al., 1989, supra).

[0189] polynucleotides

[0190] The present invention also relates to polynucleotides encoding the polypeptides of the present invention. In one embodiment, the polynucleotides encoding the polypeptides of the present invention have been isolated.

[0191] The technology for separating or cloning polynucleotides is known in the art and includes separating from genomic DNA or cDNA or its combination. Cloned DNA fragments with common structural features can be detected, for example, by using the antibody screening of well-known polymerase chain reaction (PCR) or expression library, and it is realized that polynucleotides are cloned from genomic DNA. See, for example, Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification programs such as ligase chain reaction (LCR), ligation activated transcription (LAT) and amplification (NASBA) based on polynucleotides can be used. These polynucleotides can be cloned from bacterial strains or Pseudomonas or related organisms of Bacillus, and therefore, for example, can be allelic variants or species variants of the polynucleotide polypeptide coding region.

[0192] Modification of the polynucleotide encoding the polypeptide of the present invention may be necessary for synthesizing a polypeptide substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to a non-naturally occurring form of the polypeptide. These polypeptides may be different from the polypeptides isolated from their natural sources in a certain engineered manner, such as variants different in terms of specific activity, thermostability, optimal pH, etc. These variants can be based on polynucleotides presented in the form of mature polypeptide coding sequences (e.g., subsequences thereof) of SEQ ID NO: 1 or SEQ ID NO: 18, and / or by introducing nucleotide substitutions that do not change the amino acid sequence of the polypeptide, but correspond to the codon usage of the host organism intended for producing the enzyme, or by introducing nucleotide substitutions that may produce different amino acid sequences. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107.

[0193] Nucleic acid constructs

[0194] The present invention also relates to nucleic acid constructs comprising polynucleotides of the present invention, said nucleic acid constructs being operably linked to one or more control sequences that direct the expression of the coding sequence in an expression host. Preferably, the expression is accomplished in a suitable host cell under conditions compatible with the control sequences.

[0195] The polynucleotide can be manipulated in many ways to facilitate expression of the polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to its insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art. In a preferred embodiment, the polynucleotide encodes an alanine amino acid that precedes the sequence encoding the mature phospholipase C. In another embodiment, the mature phospholipase C polypeptide comprises an N-terminal sequence beginning with the amino acids WSA, and the polypeptide expressed from the polynucleotide will produce an N-terminal sequence beginning with the amino acids AWSA.

[0196] The control sequence can be a promoter, i.e., a polynucleotide that is recognized by the host cell to express a polynucleotide encoding a polypeptide of the present invention. The promoter comprises transcriptional control sequences that mediate expression of the polypeptide. The promoter can be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant, truncated, and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0197] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in bacterial host cells are promoters obtained from the following genes: Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology). coli lac operon, the E. coli trc promoter (Egon et al., 1988, Gene 69:301-315), the Streptomyces coelicolor agarohydrolase gene (dagA), and a prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), and the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Additional promoters are described in "Useful proteins from recombinant bacteria," Gilbert et al., 1980, Scientific American, 242:74-94; and Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0198] The control sequence can also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the polypeptide. Any terminator that works in the host cell can be used in the present invention.

[0199] Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0200] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene, which increases the expression of the gene.

[0201] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

[0202] The control sequence can also be a leader, a non-translated mRNA region that is important for translation in the host cell. The leader is operably linked to the 5' end of the polynucleotide encoding the polypeptide. Any leader that works in the host cell can be used.

[0203] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that functions in the host cell may be used.

[0204] The control sequence can also be a signal peptide coding region that encodes a signal peptide that is connected to the N-terminus of the polypeptide and directs the polypeptide to enter the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide itself can be contained in a signal peptide coding sequence that is naturally connected to the coding sequence segment of the coded polypeptide in the translation reading frame. Alternatively, the 5' end of the coding sequence can contain a signal peptide coding sequence that is exogenous to the coding sequence. In the case where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, the exogenous signal peptide coding sequence can simply replace the natural signal peptide coding sequence to enhance the secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide to enter the secretory pathway of the host cell can be used.

[0205] Effective signal peptide coding sequences for bacterial host cells are those obtained from the genes for Bacillus sp. NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prs A. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

[0206] In another embodiment, the control sequence is a propeptide coding sequence of a propeptide encoding the N-terminal portion of a polypeptide. The polypeptide generated is referred to as a proenzyme (proenzyme) or propolypeptide (or in some cases is referred to as a zymogen (zymogen)). Propolypeptide is normally inactive and can be converted into an active polypeptide by catalytic cleavage or autocatalytic cleavage from the propeptide of propolypeptide. The propeptide coding sequence can be obtained from the following genes: subtilis alkaline protease (aprE), subtilis neutral protease (nprT), thermophilic myceliophthora laccase (WO 95 / 33836), miehei mucor aspartic protease and saccharomyces cerevisiae α-factor.

[0207] Where both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is positioned immediately adjacent to the N-terminus of the polypeptide and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[0208] It may also be desirable to add regulatory sequences that regulate the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that turn the expression of the gene on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operon systems. Other examples of regulatory sequences are those that allow gene amplification.

[0209] expression vector

[0210] The present invention still further relates to the recombinant expression vector that comprises polynucleotide of the present invention, promotor and transcription and translation termination signal.Different Nucleotide and control sequence can be linked together to produce recombinant expression vector, and this recombinant expression vector can comprise one or more convenient restriction enzyme sites to allow to insert or replace the polynucleotide of encoding this polypeptide at these sites.Alternately, this polynucleotide can be expressed by inserting this polynucleotide or the nucleic acid construct that comprises this polynucleotide into the appropriate vector for expression.When producing this expression vector, this encoding sequence is located in this vector, makes this encoding sequence operably connected with this appropriate control sequence for expression like this.

[0211] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be readily subjected to recombinant DNA procedures and that can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0212] The carrier can be an autonomous replicating vector, that is, a carrier existing as an extrachromosomal entity, which replicates independently of chromosomal replication, for example, a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The carrier can comprise any element in order to ensure self-replication. Alternatively, the carrier can be such a carrier that, when introduced into the host cell, is integrated into the genome and replicates with one or more chromosomes into which it has been integrated. In addition, a single vector or plasmid or two or more vectors or plasmids (these vectors or plasmids contain the total DNA to be introduced into the genome of the host cell together) or a transposon can be used.

[0213] The vector preferably contains one or more selectable markers that allow for easy selection of transformed, transfected, transduced, etc. cells. A selectable marker is a gene whose product confers biocide or viral resistance, heavy metal resistance, prototrophy of auxotrophs, etc.

[0214] Examples of bacterial selectable markers are the Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance, such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance.

[0215] The selectable marker may be a dual selectable marker system as described in WO 2010 / 039889. In one aspect, the dual selectable marker is the hph-tk dual selectable marker system.

[0216] The vector preferably contains one or more elements that permit integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0217] For being integrated into the host cell genome, the vector can rely on the polynucleotide sequence encoding the polypeptide or any other element of the vector for being integrated into the genome by homologous or non-homologous recombination. Alternatively, the vector can include other polynucleotides for guiding the integration of one or more accurate positions of one or more chromosomes in the host cell genome by homologous recombination. In order to increase the possibility of integration at the accurate position, these integration elements should include a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs and 800 to 10,000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to improve the possibility of homologous recombination. These integration elements can be any sequence homologous to the target sequence in the genome of the host cell. In addition, these integration elements can be non-coding polynucleotides or encoding polynucleotides. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

[0218] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicator that mediates autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0219] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and the origins of replication of plasmids pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus.

[0220] More than one copy of a polynucleotide of the present invention can be inserted into a host cell to increase production of a polypeptide. Increased copy numbers of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide, wherein cells containing amplified copies of the selectable marker gene, and thus additional copies of the polynucleotide, can be selected by culturing the cells in the presence of an appropriate selective agent.

[0221] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, eg, Sambrook et al., 1989, supra).

[0222] host cells

[0223] The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention. Preferably, the polynucleotide is heterologous, meaning that it is not naturally present in the host cell. A construct or vector comprising the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector, as described earlier. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication. The choice of host cell depends largely on the gene encoding the polypeptide and its source.

[0224] The host cell can be any cell useful for recombinantly producing the polypeptide of the present invention, such as a prokaryotic cell or a eukaryotic cell. In a preferred embodiment, the host cell is a recombinant host cell that does not exist in nature.

[0225] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include but are not limited to Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Ocean Bacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include but are not limited to Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silene Bacillus, Neisseria, Pseudomonas, Salmonella and Ureaplasma.

[0226] The bacterial host cell can be any Bacillus cell, including but not limited to: Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus laurens, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0227] The bacterial host cell may also be any Streptococcus cell, including but not limited to Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0228] The bacterial host cell can also be any Streptomyces cell, including but not limited to Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0229] Introduction of DNA into Bacillus cells can be accomplished by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829; or Dubnau and Davidoff-Abelson, 1990). Davidoff-Abelson, 1971, J. Mol. Biol. 56:209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). Introduction of DNA into E. coli cells can be achieved by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). Introduction of DNA into Streptomyces cells can be achieved by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. Praha 49:399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294). Introduction of DNA into Pseudomonas cells can be achieved by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64:391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:51-57).DNA is introduced into Streptococcus cells and can be achieved by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0230] Generation method

[0231] Synthetic genes based on the nucleotide sequence identified as SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO: 31 can be obtained from a number of vendors, such as Gene Art (GENEART BioPark, Josef-Engert-Str. 11, 93053 Regensburg, Germany) or DNA 2.0 (DNA2.0, 1430 O'Brien Drive, Suite E, Menlo Park, CA 94025, USA). The synthetic gene can be designed to coordinate additional DNA sequences, such as restriction sites or homologous recombination regions, to facilitate cloning into an expression vector as described above and expression in a host cell as described above, for example, in Bacillus subtilis.

[0232] One aspect of the present invention relates to a method for producing a phospholipase C polypeptide in a bacterial host, particularly a Bacillus host, more preferably a Bacillus subtilis or Bacillus licheniformis host, wherein the phospholipase C coding sequence encodes an alanine before the predicted N-terminal amino acid of the phospholipase C polypeptide. Preferably, the phospholipase C is a PC and PE-specific phospholipase C polypeptide. This results in a mature phospholipase C polypeptide having an N-terminal alanine as exemplified in SEQ ID NO:20 and SEQ ID NO:30. Preferably, the wild-type mature sequence of phospholipase C has an N-terminal sequence beginning with WSA. Without being bound by theory, the inventors believe that this additional alanine protects the N-terminal sequence of the mature phospholipase C sequence, e.g., amino acid WSA, from protease activity in the host cell.

[0233] The present invention also relates to a method for producing a phosphatidylinositol phospholipase C polypeptide of the present invention, comprising (a) cultivating a cell that produces the polypeptide in its wild-type form under conditions conducive to production of the polypeptide; and optionally (b) recovering the polypeptide. In one aspect, the cell is a Pseudomonas cell.

[0234] The present invention also relates to methods for producing the PC and PE-specific phospholipase C polypeptides of the present invention, comprising (a) cultivating a cell that produces the polypeptide in its wild-type form under conditions conducive to production of the polypeptide; and optionally (b) recovering the polypeptide. In one aspect, the cell is a Bacillus cell.

[0235] The present invention also relates to methods of producing a polypeptide of the present invention, comprising: (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide.

[0236] These host cells are cultivated in a nutrient medium suitable for producing the polypeptide using methods known in the art. For example, it is possible to culture by shaking a flask in a suitable nutrient medium and under conditions allowing expression and / or separation of the polypeptide, or to culture cells in a laboratory or industrial fermentor tank using a small or large-scale fermentation (comprising continuous, in batches, fed-batch, or solid-state fermentation). The culture is performed using a program known in the art in a suitable nutrient medium comprising a carbon source and a nitrogen source and an inorganic salt. Suitable nutrient medium can be obtained from commercial suppliers or can be prepared according to disclosed composition (for example, in the catalogue of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be directly recovered from the nutrient medium. If the polypeptide is not secreted, it can be recovered from the cell lysate.

[0237] Polypeptides having phospholipase C activity can be detected using methods known in the art, see the "Phospholipase Activity Assay" section below. These detection methods include, but are not limited to, the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate, for example, the P-NMR assay described in Example 5 or the liquid chromatography coupled to a triple quadrupole mass spectrometer (LC / MS / MS) or phosphatidylcholine plate assay described in Example 7.

[0238] The polypeptide can be recovered using methods known in the art. For example, the polypeptide can be recovered from the nutrient medium by conventional procedures, including but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. In one aspect, the fermentation broth containing the polypeptide is recovered.

[0239] The polypeptide can be purified to obtain a substantially pure polypeptide by a variety of procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, eds., VCH Publishers, New York, 1989).

[0240] In an alternative aspect, the polypeptide is not recovered, but rather a host cell of the invention expressing the polypeptide is used as a source of the polypeptide.

[0241] Phospholipase activity assay

[0242] The invention provides polypeptides (e.g., enzymes, antibodies) separated, synthesized or recombined with any combination of phospholipase activity or multiple phospholipase activities, and nucleic acids encoding them. If the polypeptide has phospholipase activity and within the scope of the present invention, any of many phospholipase activities as known in the art can be used to determine so. Conventional protocols for determining phospholipase A, B, D and C are well known in the art.

[0243] Exemplary activity assays include turbidity assays, methylumbelliferylphosphocholine (fluorescence) assays, aplex red (fluorescence) phospholipase assays, thin layer chromatography assays (TLC), cell lysis assays, and p-nitrophenylphosphocholine assays. Using these assays, polypeptides, peptides, or antibodies can be rapidly screened for phospholipase activity.

[0244] A plate assay with a substrate comprising agar can be used to determine phospholipase activity. The assay is performed as follows. Plates were cast by mixing 5 ml of 2% agarose (Litex HSA 1000) prepared by mixing and boiling for 5 minutes in a buffer (100 mM HEPES and 100 mM citrate with pH adjusted from pH 3.0 to pH 7.0) followed by cooling to approximately 60°C and 5 ml of substrate (L-alfa phosphatidylcholine, 95% from soy (Avanti 441601) or L-α-phosphatidylinositol from soy (Avanti 840044P) for PI-specificity or L-α-phosphatidylethanolamine from soy (Avanti 840024P) dispersed in water (MilliQ) at 60°C for 1 minute using an Ultra Turrax for PC-specificity), gently mixing into 7 cm diameter Petri dishes and cooling to room temperature before punching wells with a diameter of approximately 3 mm by vacuum. Ten microliters of purified enzyme diluted to 0.4 mg / ml was added to each well before the plates were sealed by parafilm and placed in an incubator for 48 hours at 55° C. Plates were photographed regularly.

[0245] Turbidimetric assays for determining phospholipase activity are described, for example, in Kauffmann (2001) "Conversion of Bacillus thermocatenulatus lipase into an efficient phospholipase with increased activity towards long-chain fatty acyl substrates by directed evolution and rational design," Protein Engineering 14:919-928; Ibrahim (1995) "Evidence implicating phospholipase as a virulence factor of Candida albicans," Infect. Immun. 63:1993-1998.

[0246] For example, methylumbelliferyl (fluorescent) phosphorylcholine for determining phospholipase activity is described in Goode (1997) "Evidence for cellsurface internal phospholipase activity in ascidian eggs," Develop. Growth Differ. 39:655-660; Diaz (1999) "Direct fluorescence-based lipase activity assay," BioTechniques 27:696-700.

[0247] The Applex Red (fluorescent) phospholipase assay for determining phospholipase activity is available as a kit, for example, the Applex Red Phosphatidylcholine-Specific Phospholipase Assay Kit from Molecular Probes, Inc. (Eugene, OR) is used to detect phosphatidylcholine-specific phospholipase according to the manufacturer's instructions.

[0248] Fluorescence was measured in a fluorescence microplate reader using excitation at 560±10 nm and fluorescence detection at 590±10 nm. The assay was sensitive at very low enzyme concentrations.

[0249] For example, thin layer chromatography assays (TLC) for determining phospholipase activity are described in Reynolds (1991) Methods in Enzymol. 197:3-13; Taguchi (1975) "Phospholipase from Clostridium novyi type A.I," Biochim. Biophys. Acta 409:75-85. Thin layer chromatography (TLC) is a widely used technique for detecting phospholipase activity. Various modifications of this method have been used to extract phospholipids from aqueous assay mixtures. In some PLC assays, the hydrolysis is stopped by adding chloroform / methanol (2:1) to the reaction mixture. Unreacted starting material and diacylglycerol are extracted into the organic phase and can be fractionated by TLC, while the head group products remain in the aqueous phase. For more accurate measurement of phospholipid digestion, radiolabeled substrate can be used (referring to, for example, Reynolds (Reynolds) (1991) Enzymology, 197:3-13). The ratio of product and reactant can be used to calculate the actual molar quantity of the substrate that enzyme unit time hydrolysis is carried out. If all components are extracted equally, any loss will affect all components equally in the extraction. Chloroform / methanol / water (65:25:4) can be used as solvent system, realize the separation of phospholipid enzymolysis product (referring to, for example, (Taguchi) (1975) Biochemistry and Biophysics Acta, 409:75-85) by silica gel TLC.

[0250] For example, the p-nitrophenylphosphorylcholine assay for determining phospholipase activity is described in Korbsrisate (1999) J. Clin. Microbiol. 37:3742-3745; Berka (1981) Infect. Immun. 34:1071-1074. The assay is based on the enzymatic hydrolysis of the substrate analog p-nitrophenylphosphorylcholine to release the yellow chromogenic compound p-nitrophenol, which is detectable at 405 nm. This substrate is convenient for high-throughput screening. Similar assays using substrates can also be applied to other phospholipid groups, for example, using p-nitrophenylphosphorylinositol or p-nitrophenylphosphorylethanolamine.

[0251] Based on the lysis of red blood cells, the cell lysis assay can detect phospholipases with cell lytic activity. Toxic phospholipases can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, causing cell lysis. See, for example, Titball (1993) Microbiol. Rev. 57: 347-366.

[0252] The Examples section of this application describes additional assays, such as 31 P-NMR and triple quadrupole mass spectrometer coupled to liquid chromatography (LC / MS / MS).

[0253] Composition

[0254] The present invention also relates to compositions comprising the PI-specific PLC polypeptides of the present invention, preferably with additional components. The PI-specific PLC polypeptides of the present invention include a) a polypeptide comprising an amino acid sequence selected from the group consisting of: i) amino acid residues 26-322 of SEQ ID NO:2 or amino acid residues 1-298 of SEQ ID NO:3; ii) amino acid residues 26-323 of SEQ ID NO:5 or amino acid residues 1-299 of SEQ ID NO:6; iii) amino acid residues 26-323 of SEQ ID NO:8 or amino acid residues 1-299 of SEQ ID NO:9; iv) amino acid residues 26-323 of SEQ ID NO:11 or amino acid residues 1-296 of SEQ ID NO:12; v) amino acid residues 26-322 of SEQ ID NO:14 or amino acid residues 1-298 of SEQ ID NO:15; vi) amino acid residues 26-322 of SEQ ID NO:17; and vii) amino acid residues 26-323 of SEQ ID NO:8 or amino acid residues 1-299 of SEQ ID NO:9. or b) a polypeptide comprising an amino acid sequence that is at least 75% identical to one of the amino acid sequences in a); or c) a functional fragment of a) or b).

[0255] The present invention also relates to compositions comprising the PC, PE-specific PLC polypeptides of the present invention, preferably with additional components.

[0256] The present invention also relates to compositions comprising a mixture of a PI-specific PLC of the present invention and one or more additional phospholipase activities selected from the group consisting of PLA1, PLA2, PLC and PLD.

[0257] The present invention also relates to a composition comprising a mixture of phosphatidylinositol phospholipase C from Pseudomonas and PC and PE-specific phospholipase C polypeptides. A preferred composition of the present invention comprises a PI-specific PLC polypeptide of the present invention in combination with a PC and PE-specific PLC polypeptide. Preferably, the PC and PE-specific PLC are selected from Purifine or the mature polypeptide of SEQ ID NO: 19, 20, 22, 24, 25, 27, 29, 30, 32 or 38. The PC and PE-specific PLC can also be selected from a sequence having at least 80%, 85%, 90%, 95% or 98% identity to the mature polypeptide of SEQ ID NO: 19, 20, 22, 24, 25, 27, 29, 30, 32 or 38, and having PC and PE-specificity. In alternative embodiments of the invention, the composition comprises a PI-specific PLC polypeptide of the invention bound to a PLC specific for PA or PC or PE; or PE and PA; or PC and PA; or PC and PE and PA; or any combination thereof.

[0258] Phospholipid hydrolase of the present invention can be formulated with the component that is selected from lower group, and this group is made up of the following: buffer agent, inorganic salt, solvent, inert solid and composition thereof.For example, suitable buffer system is to make under pH 2 to 10 with the concentration between 0.01M and 1M from the aqueous solution of salt or organic acid, amino acid, phosphate, amine or ammonia.Preferably, use the hydrochloride of alkali metal salt of citric acid, acetate, glycine and / or tris (hydroxymethyl) amine and ammonia of pH 4 to 8 of 0.1M to 0.2M.Preferably, Phospholipid hydrolase is dissolved in aqueous buffer solution, as glycine buffer, citric acid buffer etc.It has been found that the buffer that contains citrate is very suitable, is sodium citrate buffer specifically, preferably under neutral pH.

[0259] The compositions of the present invention can include a phospholipase of the present invention that is fixed to a solid support. The solid support for the present invention includes a gel. Some examples of gels include agarose, gelatin, glutaraldehyde, chitosan-treated glutaraldehyde, albumin-glutaraldehyde, chitosan-xanthan gum, toyopearl gel (polymer gel), alginate, alginate-polylysine, carrageenan, agarose, glyoxyl agarose, magnetic agarose, dextran agarose, poly (aminomethanesulfonic acid) hydrogel, BSA-PEG hydrogel, phosphorylated polyvinyl alcohol (PVA), monoethylated-N-aminoethyl (MANA), amino, or any combination thereof. Another solid support for the present invention is a resin or polymer. Some examples of resins or polymers include cellulose, acrylamide, nylon, rayon, polyester, anion exchange resin, AMBERLITE TM XAD-7, AMBERLITE TMXAD-8, AMBERLITE TM IRA-94, AMBERLITE TM IRC-50, polyvinyl alcohol, polypropylene, polymethacrylate, or any combination thereof. Another type of solid support used in the present invention is a ceramic product. Some examples include non-porous ceramics, porous ceramics, SiO2, AhO3. Another type of solid support used in the present invention is glass. Some examples include non-porous glass, porous glass, aminopropyl glass, or any combination thereof. Another type of solid support that can be used is a microelectrode. One example is polyethyleneimine-coated magnetite. Graphite particles can be used as a solid support. Other exemplary solid supports used to practice the present invention include diatomaceous earth products and silicates. Some examples include Diatomaceous earth and SILASORB TM ,and Synthetic calcium and magnesium silicates.

[0260] Some examples of the method for immobilizing enzymes include, for example, electrostatic droplet production, electrochemical methods, by adsorption, by covalent bonding, by crosslinking, by chemical reaction or method, by encapsulation, by trapping, by calcium alginate or by poly-(2-hydroxyethyl methacrylate). Similar methods are described in the following: Enzymology, Immobilized Enzymes and Cells, Part C, 1987, Academic Press, edited by SP Colowick and N.O. Kaplan, Vol. 136; and Immobilization of enzymes and cells, 1997, Humana Press, edited by GF Bickerstaff, Series: Biotechnology Methods, edited by JM Walker.

[0261] In another embodiment, the composition can include multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or a transferase, e.g., an α-galactosidase, an α-glucosidase, an aminopeptidase, an amylase, a β-galactosidase, a β-glucosidase, a β-xylosidase, a carbohydrase, a carboxypeptidase, a catalase, a cellobiohydrolase, a cellulase, a chitinase, a cutinase, a cyclodextrin glucosyltransferase, a deoxyribonuclease, an endoglucanase, an esterase, a glucoamylase, an invertase, a laccase, a lipase, a mannosidase, a mutanase, an oxidase, a pectinolytic enzyme, a peroxidase, a phytase, a polyphenol oxidase, a proteolytic enzyme, a ribonuclease, a transglutaminase, or a xylanase.

[0262] The compositions can be prepared according to methods known in the art and can be in the form of liquid or dry compositions. These compositions can be stabilized according to methods known in the art.

[0263] Examples of preferred uses of the composition of the present invention are given below. The dosage of the composition and other conditions for using the composition can be determined based on methods known in the art.

[0264] use

[0265] The phospholipases or compositions of the invention can be suitable for use in methods for removing phospholipids from oils, e.g., vegetable oils, animal oils or fats, tallow, or greases.

[0266] Applications in which the phospholipases of the invention can be used include: i) degumming of oils, e.g., vegetable oils, or edible vegetable oils, or in a process comprising hydrolysis of phospholipids in the gum fraction to release embedded triglyceride oils from water degumming, ii) in a process comprising hydrolysis of phospholipids to obtain improved phospholipid emulsifiers, particularly wherein the phospholipid is lecithin, iii) in a process for improving the filterability of an aqueous solution or slurry of a carbohydrate source comprising phospholipids, iv) in a process for extracting oil, v) in a process for producing an animal feed product, vi) in a process for producing a biofuel, e.g., biodiesel, vii) in a process for producing a detergent product, and / or viii) in a process for making a baked product, the process comprising adding a phospholipase to a dough and baking the dough to make the baked product.

[0267] The phospholipids of the invention can be used in a method comprising treating a phospholipid or lysophospholipid with a phospholipase or composition of the invention. The phospholipase or composition reacts with the phospholipid or lysophospholipid to form a monoglyceride or diglyceride and a phosphate or phosphoric acid.

[0268] Degumming Phospholipid hydrolase of the present invention and combination thereof can be used for degummed oil, for example, and animal oil or fat, tallow, grease or vegetable oil, that is, in the method for reducing the phosphatide in the oil, use.This degumming process is applicable to the purification of any edible oil that comprises phosphatide, for example, and vegetable oil is such as soybean oil, rapeseed oil or sunflower oil or any other oil of mentioning under the crude oil definition.

[0269] PI-specific PLC converts phosphatidylinositol (PI) into diglyceride and phosphoinositol. PC-specific PLC converts phosphatidylcholine (PC) into diglyceride and phosphocholine. PE-specific PLC converts phosphatidylethanolamine (PE) into diglyceride and phosphoethanolamine. This diglyceride stays in the oil phase (improving oil yield) and the phosphorus-containing part is separated into the aqueous phase, wherein it is removed as a component of the heavy phase during centrifugation. The gum phase (heavy phase) can be further processed with phospholipase of the present invention or compositions to increase the hydrolysis of phospholipids in the gum fraction, to release the embedded triglyceride oil from water degumming. When the degumming method was not yet applied with phospholipase, this was particularly useful. Phospholipase of the present invention, for example, PI-specific PLC of the present invention and / or PC, PE-specific PLC, can be incorporated into water degumming or chemical or physical oil refining process. In a preferred embodiment, the phospholipase of the invention is incorporated into a water degumming process, preferably with less than 10%, 9%, 8%, 7%, 6% or 5% water, even more preferably less than 4%, 3% or 2% water, preferably at 50°C or above, even more preferably at 60°C or above.

[0270] In another preferred embodiment, the phospholipase of the invention is incorporated into a physical refining process using citric acid or phosphoric acid and sodium hydroxide to promote hydration of insoluble phospholipids and ensure an environment suitable for the enzyme with preferably less than 0.15% citric acid or phosphoric acid, even more preferably less than 0.1%, 0.09%, 0.08%, 0.07%, 0.06% or 0.05%; and less than 4%, 3% or 2% water, preferably at 50°C or above, even more preferably at 60°C or above.

[0271] In other embodiments, the degumming process is a caustic refining process or an acid refining process.

[0272] An aspect of the present invention is a method for reducing the content of phosphatide in the oil composition, the method comprising a) providing an oil composition comprising a certain amount of phosphatide, b) under the condition that is enough to make enzyme and phosphatide reaction, the oil composition is contacted with phosphatidylinositol phospholipase C (PI-specific PLC), PC and PE-specific phospholipase C, to produce diglyceride and phosphate, and c) from the oil composition, separating phosphate. In a preferred embodiment, this phosphatidylinositol phospholipase C is from Pseudomonas. In other preferred embodiments, before contacting with phospholipase, this oil is subjected to acid / alkali treatment.

[0273] In a preferred embodiment, the phosphatidylinositol phospholipase C is a) a polypeptide comprising an amino acid sequence selected from the group consisting of: i) amino acid residues 26-322 of SEQ ID NO: 2 or amino acid residues 1-298 of SEQ ID NO: 3; ii) amino acid residues 26-323 of SEQ ID NO: 5 or amino acid residues 1-299 of SEQ ID NO: 6; iii) amino acid residues 26-323 of SEQ ID NO: 8 or amino acid residues 1-299 of SEQ ID NO: 9; iv) amino acid residues 26-323 of SEQ ID NO: 11 or amino acid residues 1-296 of SEQ ID NO: 12; v) amino acid residues 26-322 of SEQ ID NO: 14 or amino acid residues 1-298 of SEQ ID NO: 15; vi) amino acid residues 26-322 of SEQ ID NO: 17; and vii) amino acid residues 26-323 of SEQ ID NO: 8 or amino acid residues 1-299 of SEQ ID NO: 9. or b) a polypeptide comprising an amino acid sequence that is at least 75% identical to one of the amino acid sequences in a); or c) a functional fragment of a) or b).

[0274] In some embodiments, the phosphatidylinositol phospholipase C has a length of 280-320 amino acid residues, such as 280-310 amino acid residues, 280-305 amino acid residues, 280-300 amino acid residues, 280-298 amino acid residues, 280-297 amino acid residues, 280-296 amino acid residues, 285-320 amino acid residues, 285-315 amino acid residues, 285-310 amino acid residues, 285-305 amino acid residues, 285-300 amino acid residues, 285-298 amino acid residues, 285-297 amino acid residues, 285-296 amino acid residues. , 290-320 amino acid residues, 290-315 amino acid residues, 290-310 amino acid residues, 290-305 amino acid residues, 290-300 amino acid residues, 290-298 amino acid residues, 290-297 amino acid residues, 290-296 amino acid residues, 295-320 amino acid residues, 295-315 amino acid residues, 295-310 amino acid residues, 295-305 amino acid residues, 295-300 amino acid residues, 295-298 amino acid residues, 255-297 amino acid residues, or 295-296 amino acid residues in length.

[0275] According to other embodiments, the PC and PE-specific phospholipase C has a length of 220-280 amino acid residues, such as 220-270 amino acid residues, 220-260 amino acid residues, 220-250 amino acid residues, 220-248 amino acid residues, 220-246 amino acid residues, 220-244 amino acid residues, 225-280 amino acid residues, 225-270 amino acid residues, 225-260 amino acid residues, 225-250 amino acid residues, amino acid residues, 225-248 amino acid residues, 225-246 amino acid residues, 225-244 amino acid residues, 230-280 amino acid residues, 230-270 amino acid residues, 230-260 amino acid residues, 230-250 amino acid residues, 230-248 amino acid residues, 230-246 amino acid residues, 230-244 amino acid residues, 235-280 amino acid residues, 235-270 amino acid residues, 235-260 amino acid residues amino acid residues, 235-250 amino acid residues, 235-248 amino acid residues, 235-246 amino acid residues, 235-244 amino acid residues, 240-280 amino acid residues, 240-270 amino acid residues, 240-260 amino acid residues, 240-250 amino acid residues, 240-248 amino acid residues, 240-246 amino acid residues, 240-244 amino acid residues, 242-280 amino acid residues, 242-270 amino acid residues, 242-260 amino acid residues, 242-250 amino acid residues, 242-248 amino acid residues, 242-246 amino acid residues, 242-244 amino acid residues, 243-280 amino acid residues, 243-270 amino acid residues, 243-260 amino acid residues, 243-250 amino acid residues, 243-248 amino acid residues, 243-246 amino acid residues, 243-244 amino acid residues in length.

[0276] In yet further embodiments, the oil is contacted with:

[0277] 0.5-200 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 0.5-200 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C;

[0278] 0.5-100 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 0.5-100 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C;

[0279] 0.5-25 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 0.5-25 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C;

[0280] 0.5-15 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 0.5-15 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C; 0.5-10 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 0.5-10 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0281] 0.5-5 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 0.5-5 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0282] 1-200 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 1-200 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C;

[0283] 1-100 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 1-100 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C;

[0284] 1-25 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 1-25 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C;

[0285] 1-15 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 1-15 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C; 1-10 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 1-10 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0286] 1-5 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 1-5 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0287] 2-200 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-200 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0288] 2-100 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-100 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0289] 2-50 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-50 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0290] 2-25 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-25 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0291] 2-15 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-15 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0292] 2-10 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-10 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0293] 2-7 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-7 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C, or

[0294] 2-5 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-5 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C.

[0295] In another embodiment, the PC and PE-specific phospholipase C is a) a polypeptide comprising an amino acid sequence selected from the group consisting of: i) amino acid residues 34-278 of SEQ ID NO: 19 or amino acid residues 1-246 of SEQ ID NO: 20; ii) amino acid residues 25-283 of SEQ ID NO: 22 or amino acid residues 39-283 of SEQ ID NO: 22; iii) amino acid residues 25-283 of SEQ ID NO: 24 or amino acid residues 39-283 of SEQ ID NO: 24 or amino acid residues 1-260 of SEQ ID NO: 25; iv) amino acid residues 25-283 of SEQ ID NO: 27 or amino acid residues 39-283 of SEQ ID NO: 27; v) amino acid residues 28-289 of SEQ ID NO: 29 or amino acid residues 52-289 of SEQ ID NO: 29 or amino acid residues 1-263 of SEQ ID NO: 30; vi) amino acid residues 25-283 of SEQ ID NO: 24 or amino acid residues 39-283 of SEQ ID NO: 24 or amino acid residues 1-260 of SEQ ID NO: 25; NO:32 or amino acid residues 38-282 of SEQ ID NO:32; vii) amino acid residues 25-280 of SEQ ID NO:38 or amino acid residues 36-280 of SEQ ID NO:38; and vii) Purafine; or b) a polypeptide comprising an amino acid sequence that is at least 75% identical to one of the amino acid sequences in a); or c) a functional fragment of a) or b).

[0296] Another aspect of the invention is a method for reducing the phospholipid content of an oil composition, the method comprising: a) providing an oil composition comprising an amount of phospholipids, b) contacting the oil composition with a PC and PE-specific phospholipase C selected from the group consisting of: i) amino acid residues 34-278 of SEQ ID NO: 19 or amino acid residues 1-246 of SEQ ID NO: 20; 2) amino acid residues 25-283 of SEQ ID NO: 24, amino acid residues 39-283 of SEQ ID NO: 24, or amino acid residues 1-260 of SEQ ID NO: 25; 3) amino acid residues 25-283 of SEQ ID NO: 27 or amino acid residues 39-283 of SEQ ID NO: 27; 4) amino acid residues 28-289 of SEQ ID NO: 29, or amino acid residues 52-289 of SEQ ID NO: 29, or amino acid residues 53-54 of SEQ ID NO: 29; IDNO:30 amino acid residues 1-263; ii) a polypeptide comprising an amino acid sequence having at least 75% identity with one of the amino acid sequences in i); or iii) a functional fragment of i) or ii) to produce a diglyceride and a phosphate ester, and c) separating the phosphate ester from the oil composition.

[0297] Phospholipids in oil are usually measured as "phosphorus content" in parts per million. Table 1 lists typical amounts of phospholipids present in major oilseed crops, and the distribution of each functional group as a percentage of the phospholipids present in the oil.

[0298] Table 1: Typical levels and phospholipid profiles of common oilseeds

[0299] soybean oil rapeseed oil sunflower oil Phosphorus (ppm) 400-1500 200-900 300-700 PC% 12-46 25-40 29-52 PE% 8-34 15-25 17-26 PA% 17-26 10-20 15-30 PI% 2-15 2-25 11-22

[0300] The enzymes and methods of the invention can be used to achieve more complete degumming of high phosphorus oils, e.g., oils having greater than 200 ppm phosphorus, preferably greater than 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, even more preferably the oil contains greater than 1000 ppm phosphorus.

[0301] Preferably, the oil comprises phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI). Preferably, the oil comprises more than 50ppm phosphorus derived from phosphatidylinositol (PI), more preferably it comprises more than 75ppm, 100ppm, 125ppm PI, even more preferably it comprises more than 150ppm, preferably it comprises more than 175ppm phosphorus derived from PI. Preferably, the oil comprises more than 100ppm phosphorus derived from phosphatidylcholine (PC), more preferably it comprises more than 150ppm, 200ppm, 250ppm PC, even more preferably it comprises more than 300ppm, preferably it comprises more than 400ppm phosphorus derived from PC. Preferably, the oil comprises more than 75ppm phosphorus derived from phosphatidylethanolamine (PE), more preferably it comprises more than 100ppm, 125ppm, 150ppm PE, even more preferably it comprises more than 200ppm, preferably it comprises more than 300ppm phosphorus derived from PE.

[0302] In a preferred embodiment, this oil is an edible oil. More preferably, this edible oil is selected from rice bran, rapeseed, palm, peanut and other nuts, soybean, corn, rape and sunflower oil. Phospholipid hydrolase of the present invention can be used for any " degumming " process, comprise water degumming, ALCON oil degumming (for example, for soybean), safinco degumming, " super degumming ", UF degumming, TOP degumming, single degumming, dry degumming and ENZYMAX TMDegumming. See, for example, WO 2007 / 103005, US 2008 / 0182322, US 6,355,693, US 6,162,623, US 6,103,505, US 6,001,640, US 5,558,781, and US 5,264,367, which describe degumming methods, in which the phospholipases of the present invention can be used. Various "degumming" processes incorporated by the methods of the present invention are described below, in Bockisch, M. (1998) Fats and Oils Handbook, The Extraction of Vegetable Oils (Chapter 5), 345-5445, AOCS Press, Champaign, Illinois. For example, the phospholipases of the present invention can be used in industrial applications of enzymatic degumming of triglyceride oils as described in EP 513 709. In another embodiment, the oil is selected from crude oil, water degummed oil, caustic refined oil and acid degummed oil. The water degumming of crude oil or fat can be achieved by fully mixing hot water and hot oil or fat with a temperature between 50 ℃ and 90 ℃ for 30 to 60 minutes. This method is used to remove the phosphatide that can be hydrated. Meanwhile, acid treatment can be carried out before enzymatic degumming, wherein the acid used is selected from the group consisting of phosphoric acid, acetic acid, citric acid, tartaric acid, succinic acid and its mixture, specifically preferably using the processing of citric acid and phosphoric acid. This acid treatment preferably carries out a neutralization step subsequently to adjust the pH between about 4.0 and 7.0, more preferably from 4.5 to 6.5, preferably using NaOH or KOH. This acid treatment is used to chelate the metal bound to phosphatide, thereby making a more hydratable form. Preferably, after the water degumming or acid treatment of oil, phospholipase as described herein is added. The degumming step can also be performed on crude oils or fats, i.e., oils or fats that have not been previously water-degummed or acid-treated, using a phospholipase as described herein.

[0303] In one aspect, the present invention provides a method for producing a soluble solid under low water conditions, for example, between about 0.1% and 20%

[0304] In one embodiment, the present invention relates to a method for enzymatic degumming of oily matter. The method comprises the steps of: separating the oily matter from the oil phase in a centrifugal process and separating the heavy phase from the oil phase. The improved separation of these phases can result in more effectively removing phosphatide from oil, including hydratable and non-hydratable phosphatide. On the one hand, this can produce a gum fraction comprising the neutral oil (triglyceride) less than that entrained, thereby improving the gross output value of oil in the degumming process. On the one hand, use phospholipase of the present invention, for example, PI-specific-PLC and / or PC, PE-specific PLC to process oil, to reduce glue quality and to increase the neutral oil results by the oil embedding that reduces. On the one hand, use phospholipase of the present invention, for example, polypeptide with PLC activity, to produce diacylglycerol (DAG) and to help form oil phase.

[0305] The phospholipase treatment can be carried out by dispersing an aqueous phospholipase solution, preferably as droplets having a mean diameter lower than 10 microns. The amount of water is preferably 0.5%-5% relative to the weight of the oil. An emulsifier can be optionally added. Mechanical agitation can be used to maintain the emulsion. A high shear mixer can be used to complete stirring at a tip speed higher than 1400 cm / s.

[0306] In certain embodiments, the oil degumming method that is applicable to comprises a) the aqueous solution of acid is mixed with oil to obtain the acidic mixture with the pH of about 1 to 4, b) alkali is mixed with the acidic mixture to obtain the mixture of the reaction with the pH of about 6-9, and c) with enzyme of the present invention, the mixture of reaction is degummed, to obtain the oil of degumming.In certain embodiments, step a) and / or b) mixing produces a kind of emulsion, and this emulsion comprises the aqueous phase of average droplet size between about 15 microns to about 45 microns.In certain embodiments, step a) and / or b) mixing produces a kind of emulsion, and this emulsion comprises at least about 60% aqueous phase by volume, the size of droplet size between about 15 microns to about 45 microns, and wherein the aqueous phase percentage ratio is based on the cumulative volume of aqueous phase.Can use any those skilled in the art to be considered as suitable acid in the method that this provides.In certain embodiments, this acid is selected from lower group, and this group is made up of the following: phosphoric acid, acetic acid, citric acid, tartaric acid, succinic acid and its composition.Can use any those skilled in the art to be considered as suitable acid in the method that this provides. In certain embodiments, the base is selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium silicate, sodium carbonate, calcium carbonate, and combinations thereof.

[0307] In a preferred embodiment, the phospholipase treatment can be carried out at a pH in the range of about 4.0 to 7.0, preferably from 4.5 to 6.5. The pH is measured in the emulsion or at the interface between the oil and the aqueous solution. Suitable temperatures are generally 30°C to 80°C. In a preferred embodiment, the temperature of the oil is between 50°C and 70°C, more preferably between 55°C and 65°C and most preferably between 50°C and 60°C. In other preferred embodiments, the temperature of the oil is between 60°C and 80°C, more preferably between 65°C and 75°C and most preferably between 67°C and 72°C.

[0308] The reaction times is typically 1-12 hour (for example, 1-6 hour or 1-3 hour, most preferably the reaction times is between 1.5 and 4 hours, even more preferably between 1.5 and 2 hours). The enzyme dosage that is fit to is normally 0.1-10mg / liter (for example, 0.5-5mg / liter). The phospholipase treatment can be carried out in batches, for example, in the tank with stirring, or can be carried out continuously, for example serial stirred tank reactor. The phospholipase treatment can be carried out after separating aqueous phase and oil phase. Can be by traditional means, for example centrifugal carry out this separation. When using liquid lipase, this water will comprise phospholipase, and this enzyme can be reused, to improve this method economy.

[0309] In a preferred embodiment of the present invention, the treatment reduces the total phosphorus content of the oil to less than 200 ppm, preferably less than 100 ppm, less than 50 ppm, less than 40 ppm, 30 ppm, 20 ppm, 15 ppm, more preferably less than 10 ppm, less than 9 ppm, less than 8 ppm, less than 7 ppm, less than 6 ppm, and most preferably less than 5 ppm.

[0310] In addition to the phospholipases of the present invention, other enzymes can be used in the degumming process outlined above. In a preferred embodiment, the other enzyme is a polypeptide having phospholipase A1, A2 and / or B activity. A suitable polypeptide having phospholipase A1 activity can be LECITASE ULTRA available from Novozymes.

[0311] Phospholipid emulsifier Phospholipid hydrolase of the present invention can be used for partial hydrolysis of phospholipids, preferably lecithin, to obtain improved phospholipid emulsifiers. The application is further described in Ullmann's Encyclopedia of Industrial Chemistry (Ullmann's Encyclopedia of Industrial Chemistry) (publisher: VCH Weinheim (Weinheim) (1996)), JP Patent 2794574, and JP-B 6-087751.

[0312] filterThe phospholipases of the present invention can be used to improve the filterability of aqueous solutions or slurries of carbohydrate sources by treating them with the phospholipases. This method is particularly suitable for solutions containing slurries of starch hydrolysates, especially wheat starch hydrolysates, which are difficult to filter and produce a cloudy filtrate. The treatment can be performed using methods similar to those described in EP 219,269 (CPC International).

[0313] Animal feed The phospholipases of the invention can be used in a method for producing animal feed, the method comprising mixing the phospholipases with a feed substrate comprising at least one phospholipid. This can be accomplished similarly to EP 743 017.

[0314] biodiesel Phospholipases of the present invention can be used in combination with one or more lipolytic enzymes to convert fats and oils into fatty acid alkyl esters while simultaneously achieving degumming in the same process. For example, such a method is described in US 8,012,724.

[0315] detergent : The phospholipase of the invention can be added to a detergent composition and thus used as an ingredient of a detergent composition.

[0316] The detergent compositions can be formulated, for example, as hand or machine laundry additive compositions, including laundry additive compositions suitable for pretreatment of soiled fabrics and fabric softener compositions with added rinse agents, or as detergent compositions for use in general household hard surface cleaning operations, or as detergent compositions for hand or machine dishwashing operations.

[0317] Baking The phospholipases of the invention can be used in the production of dough and baked products from dough, as well as in the production of baking compositions and baking additives.

[0318] The dough typically includes wheat meal or wheat flour and / or other types of meal, flour or starch such as corn meal, corn starch, rye meal, rye starch, oat flour, oat meal, soy flour, sorghum meal, sorghum flour, potato meal, potato flour or potato starch.

[0319] The dough may be fresh, frozen or par-baked.

[0320] The dough is typically a leavened dough or a dough that is to be subjected to leavening. The dough can be leavened in various ways, such as by adding a chemical leavening agent (e.g., sodium bicarbonate) or by adding a fermentation agent (fermented dough), but preferably the dough is leavened by adding a suitable yeast culture, such as a culture of Saccharomyces cerevisiae (baker's yeast), for example a commercially available strain of Saccharomyces cerevisiae.

[0321] The dough may also include other traditional dough ingredients, for example: protein, such as milk powder, gluten and soy; egg (or whole egg, or egg yolk or egg white); oxidizing agent, such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate; amino acid, such as L-cysteine; sugar; salt, such as sodium chloride, calcium acetate, sodium sulfate or calcium sulfate.

[0322] The dough may include fat (triglycerides), such as granulated fat or shortening, but the invention is particularly applicable to doughs to which less than 1% by weight of fat has been added, and is particularly applicable to doughs made without added fat.

[0323] The dough may further include an emulsifier such as mono- or diglycerides, diacetyl tartaric acid esters of mono- or diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetate esters of monoglycerides, polyoxyethylene stearate, or lysolecithin.

[0324] Dough is any type of baked product that can be prepared from dough, which can have soft or crisp properties and can be white, light or dark. Examples are bread (particularly white, whole-meal or rye bread), typically in the form of loaves or rolls, baguettes, pita bread, tortillas, cakes, pancakes, biscuits, waffles, cookies, pie crusts, crispbread, steamed buns, pizza, etc.

[0325] The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0326] project

[0327] 1. A method for reducing the phospholipid content in an oil composition, the method comprising

[0328] a) providing an oil composition comprising a certain amount of phospholipids,

[0329] b) contacting the oil composition with a phosphatidylinositol phospholipase C and PC and PE-specific phospholipase C under conditions sufficient for the enzyme to react with the phospholipid to produce a diacylglycerol and a phosphate; and,

[0330] c) separating the phosphate ester from the oil composition.

[0331] 2. The method according to item 1, wherein the phosphatidylinositol phospholipase C is derived from the genus Pseudomonas.

[0332] 3. The method according to item 1 or 2, wherein the oil is an edible oil.

[0333] 4. The method according to any one of the above items, wherein the oil is selected from crude oil, water degummed oil, caustic refined oil and acid degummed oil.

[0334] 5. The method according to any one of the above items, wherein the oil comprises phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI).

[0335] 6. The method of claim 5, wherein the oil comprises at least 50 ppm phosphorus derived from phosphatidylinositol (PI).

[0336] 7. The method according to any one of the above items, wherein the phosphatidylinositol phospholipase C is

[0337] a) a polypeptide comprising an amino acid sequence selected from the group consisting of:

[0338] i) amino acid residues 26-322 of SEQ ID NO: 2 or amino acid residues 1-298 of SEQ ID NO: 3;

[0339] ii) amino acid residues 26-323 of SEQ ID NO: 5 or amino acid residues 1-299 of SEQ ID NO: 6;

[0340] iii) amino acid residues 26-323 of SEQ ID NO: 8 or amino acid residues 1-299 of SEQ ID NO: 9;

[0341] iv) amino acid residues 26-323 of SEQ ID NO: 11 or amino acid residues 1-296 of SEQ ID NO: 12;

[0342] v) amino acid residues 26-322 of SEQ ID NO: 14 or amino acid residues 1-298 of SEQ ID NO: 15;

[0343] vi) amino acid residues 26-322 of SEQ ID NO: 17; and

[0344] vii) amino acid residues 28-339 of SEQ ID NO: 35; or

[0345] b) a polypeptide comprising an amino acid sequence that is at least 75% identical to one of the amino acid sequences in a); or

[0346] c) A functional fragment of a) or b).

[0347] 8. The method according to any one of the above items, wherein the PC and PE-specific phospholipase C are

[0348] a) a polypeptide comprising an amino acid sequence selected from the group consisting of:

[0349] i) amino acid residues 34-278 of SEQ ID NO: 19 or amino acid residues 1-246 of SEQ ID NO: 20;

[0350] ii) amino acid residues 25-283 of SEQ ID NO: 22 or amino acid residues 39-283 of SEQ ID NO: 22;

[0351] iii) amino acid residues 25-283 of SEQ ID NO: 24 or amino acid residues 39-283 of SEQ ID NO: 24 or amino acid residues 1-260 of SEQ ID NO: 25;

[0352] iv) amino acid residues 25-283 of SEQ ID NO:27 or amino acid residues 39-283 of SEQ ID NO:27;

[0353] v) amino acid residues 28-289 of SEQ ID NO:29 or amino acid residues 52-289 of SEQ ID NO:29 or amino acid residues 1-263 of SEQ ID NO:30;

[0354] vi) amino acid residues 21-282 of SEQ ID NO: 32 or amino acid residues 38-282 of SEQ ID NO: 32;

[0355] vii) amino acid residues 25-280 of SEQ ID NO: 38 or amino acid residues 36-280 of SEQ ID NO: 38; and

[0356] viii) Purifine; or

[0357] b) a polypeptide comprising an amino acid sequence that is at least 75% identical to one of the amino acid sequences in a); or

[0358] c) A functional fragment of a) or b).

[0359] 9. The method according to any of the preceding items, wherein the phosphatidylinositol phospholipase C is a phosphoinositide phospholipase C having a length of 280-320 amino acid residues, such as 280-310 amino acid residues, 280-305 amino acid residues, 280-300 amino acid residues, 280-298 amino acid residues, 280-297 amino acid residues, 280-296 amino acid residues, 285-320 amino acid residues, 285-315 amino acid residues, 285-310 amino acid residues, 285-305 amino acid residues, 285-300 amino acid residues, 285-298 amino acid residues, 285-297 amino acid residues, 285-296 amino acid residues. 6 amino acid residues, 290-320 amino acid residues, 290-315 amino acid residues, 290-310 amino acid residues, 290-305 amino acid residues, 290-300 amino acid residues, 290-298 amino acid residues, 290-297 amino acid residues, 290-296 amino acid residues, 295-320 amino acid residues, 295-315 amino acid residues, 295-310 amino acid residues, 295-305 amino acid residues, 295-300 amino acid residues, 295-298 amino acid residues, 255-297 amino acid residues, or 295-296 amino acid residues in length.

[0360] 10. The method according to any of the preceding items, wherein the PC and PE-specific phospholipase C is a phospholipase C having a length of 220-280 amino acid residues, such as 220-270 amino acid residues, 220-260 amino acid residues, 220-250 amino acid residues, 220-248 amino acid residues, 220-246 amino acid residues, 220-244 amino acid residues, 225-280 amino acid residues, 225-270 amino acid residues, 225-260 amino acid residues. , 225-250 amino acid residues, 225-248 amino acid residues, 225-246 amino acid residues, 225-244 amino acid residues, 230-280 amino acid residues, 230-270 amino acid residues, 230-260 amino acid residues, 230-250 amino acid residues, 230-248 amino acid residues, 230-246 amino acid residues, 230-244 amino acid residues, 235-280 amino acid residues, 235-270 amino acid residues, 2 35-260 amino acid residues, 235-250 amino acid residues, 235-248 amino acid residues, 235-246 amino acid residues, 235-244 amino acid residues, 240-280 amino acid residues, 240-270 amino acid residues, 240-260 amino acid residues, 240-250 amino acid residues, 240-248 amino acid residues, 240-246 amino acid residues, 240-244 amino acid residues, 242-280 amino acid residues, 242 -270 amino acid residues, 242-260 amino acid residues, 242-250 amino acid residues, 242-248 amino acid residues, 242-246 amino acid residues, 242-244 amino acid residues, 243-280 amino acid residues, 243-270 amino acid residues, 243-260 amino acid residues, 243-250 amino acid residues, 243-248 amino acid residues, 243-246 amino acid residues, 243-244 amino acid residues in length.

[0361] 11. The method according to any one of the above items, wherein the oil is contacted with:

[0362] 0.5-200 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 0.5-200 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C;

[0363] 0.5-100 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 0.5-100 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C;

[0364] 0.5-25 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 0.5-25 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C;

[0365] 0.5-15 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 0.5-15 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C; 0.5-10 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 0.5-10 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0366] 0.5-5 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 0.5-5 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0367] 1-200 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 1-200 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C;

[0368] 1-100 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 1-100 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C;

[0369] 1-25 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 1-25 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C;

[0370] 1-15 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 1-15 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C; 1-10 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 1-10 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0371] 1-5 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 1-5 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0372] 2-200 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-200 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0373] 2-100 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-100 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0374] 2-50 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-50 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0375] 2-25 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-25 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0376] 2-15 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-15 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0377] 2-10 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-10 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0378] 2-7 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-7 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C,

[0379] 2-5 mg enzyme protein (EP) / Kg oil of the phosphatidylinositol phospholipase C and 2-5 mg enzyme protein (EP) / Kg oil of the PC and PE-specific phospholipase C.

[0380] 12. A polypeptide having phosphatidylinositol phospholipase C activity, the polypeptide being selected from the group consisting of:

[0381] a) a polypeptide having at least 91% sequence identity to the mature polypeptide of SEQ ID NO: 2;

[0382] b) a polypeptide encoded by a polynucleotide that hybridizes under moderate stringency conditions with

[0383] i) the mature polypeptide coding sequence of SEQ ID NO: 1, or

[0384] ii) the full-length complement of (i);

[0385] c) a polypeptide encoded by a polynucleotide having at least 90% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1;

[0386] d) a variant of the mature polypeptide of SEQ ID NO: 2, the variant comprising a substitution, deletion, and / or insertion at one or more positions; and

[0387] e) A fragment of the polypeptide of (a), (b), (c), or (d), which fragment has phosphatidylinositol phospholipase C activity.

[0388] 13. The polypeptide according to item 12, which has a thermal denaturation temperature of at least 60°C, such as 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C or at least 90°C as determined by differential scanning calorimetry (DSC).

[0389] 14. The polypeptide according to item 12 or 13, wherein the denaturation temperature is determined as the apex of the denaturation peak (main endothermic peak) in the thermogram (Cp vs. T) obtained after heating a 1 mg / ml solution of the polypeptide in a buffer (50 mM Na-acetoacetate (pH 5.5), or 50 mM Hepes (pH 7)) at a constant programmed heating rate of 200 K / hr.

[0390] 15. The polypeptide according to any one of items 12 to 14, which is capable of reducing the phosphatidylinositol content of crude soybean oil by 50% or more, such as 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more, the reduction in phosphatidylinositol content being achieved after adding 100 mg enzyme protein (EP) / kg oil and incubating the oil and enzyme at 50° C. at pH 5.5 for 2 hours. 31 Confirmed by P-NMR.

[0391] 16. The polypeptide according to any one of items 12 to 15, which is capable of reducing the phosphorus content of crude soybean oil to 20 mg / kg oil or less as measured by inductively coupled plasma optical emission spectrometry (ICP-OES) after incubation of 4 mg enzyme protein / kg oil at 50° C. to 60° C. for 5 hours in a low-water system comprising 3% water based on the amount of oil.

[0392] 17. The polypeptide according to item 16, wherein the crude soybean oil comprises 80-140 ppm phosphorus present as phosphatidic acid (PA), 140-200 ppm phosphorus present as phosphatidylethanolamine (PE), 70-110 ppm phosphorus present as phosphatidic acid (PI) and 130-200 ppm phosphorus present as phosphatidylcholine; the phosphorus content is determined by 31P-NMR measurement.

[0393] 18. The polypeptide according to item 16 or 17, wherein the reduction in phosphorus content is obtained in an oil degumming process comprising the steps of:

[0394] i) optionally treating crude soybean oil with acid / base by adding 85% orthophosphoric acid solution in an amount corresponding to 0.05% (100% pure orthophosphoric acid) based on the amount of oil, mixing in an ultrasonic bath for 5 minutes, followed by incubation in a rotator for 15 minutes and neutralization with 4M NaOH base in an amount equivalent to pure orthophosphoric acid (from 0.5 to 0.15) in an ultrasonic bath for 5 minutes;

[0395] ii) adding the polypeptide to the oil in an amount of 4 mg enzyme protein / kg oil in a low-water system including 3% water based on the amount of oil, and subjecting the oil and the polypeptide to ultrasonic treatment for 5 minutes;

[0396] iii) incubating the polypeptide and oil at 50° C.-60° C. for 5 hours with stirring at 20 rpm;

[0397] iv) Centrifuge the oil and polypeptide at 700 g at 85°C for 15 minutes.

[0398] 19. The polypeptide according to any one of items 11-18, comprising, consisting of or essentially consisting of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 2 or amino acids 26 to 322 of SEQ ID NO: 2 or amino acids 1 to 298 of SEQ ID NO: 3.

[0399] 20. The polypeptide according to any one of items 12 to 19, having a length of 280-320 amino acid residues, such as 280-310 amino acid residues, 280-305 amino acid residues, 280-300 amino acid residues, 280-298 amino acid residues, 280-297 amino acid residues, 280-296 amino acid residues, 285-320 amino acid residues, 285-315 amino acid residues, 285-310 amino acid residues, 285-305 amino acid residues, 285-300 amino acid residues, 285-298 amino acid residues, 285-297 amino acid residues, 285-296 amino acid residues. or a length of 295-296 amino acid residues.

[0400] 21. A polypeptide having PC and PE specific phospholipase C activity, the polypeptide being selected from the group consisting of:

[0401] a) a polypeptide having at least 70% sequence identity to the mature polypeptide of SEQ ID NO: 19;

[0402] b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions to

[0403] i) the mature polypeptide coding sequence of SEQ ID NO: 18, or

[0404] ii) the full-length complement of (i);

[0405] c) a polypeptide encoded by a polynucleotide having at least 70% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 18;

[0406] d) a variant of the mature polypeptide of SEQ ID NO: 19, the variant comprising a substitution, deletion, and / or insertion at one or more positions; and

[0407] e) A fragment of the polypeptide of (a), (b), (c), or (d), which fragment has PC- and PE-specific phospholipase C activity.

[0408] 22. The polypeptide according to item 21, which has a thermal denaturation temperature of at least 60°C, such as 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C or at least 90°C as determined by differential scanning calorimetry (DSC).

[0409] 23. The polypeptide according to item 22, wherein the denaturation temperature is determined as the apex of the denaturation peak (main endothermic peak) in the thermogram (Cp vs. T) obtained after heating a 1 mg / ml solution of the polypeptide in a buffer (50 mM Na-acetoacetate (pH 5.5), or 50 mM Hepes (pH 7)) at a constant programmed heating rate of 200 K / hr.

[0410] 24. The polypeptide according to any one of items 21 to 23, which is capable of reducing the phosphatidylethanolamine and / or phosphatidylcholine content of crude soybean oil by 50% or more, such as 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more, the reduction in phosphatidylethanolamine and / or phosphatidylcholine content being achieved after adding 100 mg enzyme protein (EP) / kg oil and incubating the oil and enzyme at 50° C. at pH 5.5 for 2 hours. 31 Confirmed by P-NMR.

[0411] 25. The polypeptide according to any one of items 21 to 24, which is capable of reducing the phosphorus content of crude soybean oil to 20 mg / kg oil or less as measured by inductively coupled plasma optical emission spectrometry (ICP-OES) after incubation of 4 mg enzyme protein / kg oil at 50°C-60°C for 5 hours in a low-water system comprising 3% water based on the amount of oil.

[0412] 26. The polypeptide according to any one of items 24-25, wherein the crude soybean oil comprises 80-140 ppm phosphorus present as phosphatidic acid (PA), 140-200 ppm phosphorus present as phosphatidylethanolamine (PE), 70-110 ppm phosphorus present as phosphatidic acid (PI) and 130-200 ppm phosphorus present as phosphatidylcholine; the phosphorus content is determined by 31 P-NMR measurement.

[0413] 27. The polypeptide according to any one of items 24 to 26, wherein the reduction in phosphorus content and / or the reduction in phosphatidylethanolamine and / or phosphatidylcholine content is obtained in an oil degumming process comprising the following steps:

[0414] i) optionally treating crude soybean oil with acid / base by adding 85% orthophosphoric acid solution in an amount corresponding to 0.05% (100% pure orthophosphoric acid) based on the amount of oil, mixing in an ultrasonic bath for 5 minutes, followed by incubation in a rotator for 15 minutes and neutralization with 4M NaOH base in an amount equivalent to pure orthophosphoric acid (from 0.5 to 0.15) in an ultrasonic bath for 5 minutes;

[0415] ii) adding the polypeptide to the oil in an amount of 4 mg enzyme protein / kg oil in a low-water system including 3% water based on the amount of oil, and subjecting the oil and the polypeptide to ultrasonic treatment for 5 minutes;

[0416] iii) incubating the polypeptide and oil at 50° C.-60° C. for 5 hours with stirring at 20 rpm;

[0417] iv) Centrifuge the oil and polypeptide at 700 g at 85°C for 15 minutes.

[0418] 28. The polypeptide according to any one of items 21-27, comprising, consisting of or essentially consisting of SEQ ID NO: 19 or the mature polypeptide of SEQ ID NO: 19 or amino acids 34 to 278 of SEQ ID NO: 19 or amino acids 1-246 of SEQ ID NO: 20.

[0419] 29. The polypeptide according to any one of items 21 to 28, having a length of 220-280 amino acid residues, such as 220-270 amino acid residues, 220-260 amino acid residues, 220-250 amino acid residues, 220-248 amino acid residues, 220-246 amino acid residues, 220-244 amino acid residues, 225-280 amino acid residues, 225-270 amino acid residues, 225-260 amino acid residues, 225-250 amino acid residues. residues, 225-248 amino acid residues, 225-246 amino acid residues, 225-244 amino acid residues, 230-280 amino acid residues, 230-270 amino acid residues, 230-260 amino acid residues, 230-250 amino acid residues, 230-248 amino acid residues, 230-246 amino acid residues, 230-244 amino acid residues, 235-280 amino acid residues, 235-270 amino acid residues, 235-260 amino acid residues, 235-250 amino acid residues, 235-248 amino acid residues, 235-246 amino acid residues, 235-244 amino acid residues, 240-280 amino acid residues, 240-270 amino acid residues, 240-260 amino acid residues, 240-250 amino acid residues, 240-248 amino acid residues, 240-246 amino acid residues, 240-244 amino acid residues, 242-280 amino acid residues, 242-2 70 amino acid residues, 242-260 amino acid residues, 242-250 amino acid residues, 242-248 amino acid residues, 242-246 amino acid residues, 242-244 amino acid residues, 243-280 amino acid residues, 243-270 amino acid residues, 243-260 amino acid residues, 243-250 amino acid residues, 243-248 amino acid residues, 243-246 amino acid residues, 243-244 amino acid residues in length.

[0420] 30. A polynucleotide encoding the polypeptide according to any one of items 12 to 29.

[0421] 31. A nucleic acid construct or expression vector comprising the polynucleotide of item 30 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

[0422] 32. A recombinant host cell comprising the polynucleotide of item 30 operably linked to one or more control sequences that direct the production of the polypeptide.

[0423] 33. A method of producing a polypeptide as described in any one of items 12 to 29, the method comprising: cultivating a cell under conditions conducive for production of the polypeptide, the cell producing the polypeptide in its wild-type form.

[0424] 34. A method for producing a phospholipase C polypeptide in a Bacillus host, wherein the phospholipase C coding sequence encodes an alanine before the predicted N-terminal amino acid of the phospholipase C polypeptide.

[0425] 35. The method of item 34, wherein the phospholipase C polypeptide is a PC and PE-specific phospholipase C polypeptide.

[0426] 36. A method of producing the polypeptide of any one of items 12 to 29, the method comprising: culturing the host cell of item 32 under conditions conducive for production of the polypeptide.

[0427] 37. The method of any one of items 33 to 37, further comprising recovering the polypeptide.

[0428] 38. A composition comprising the polypeptide according to any one of items 12-2013.

[0429] 39. A composition comprising the polypeptide of any one of items 21-29.

[0430] 40. A composition comprising a mixture of a phosphatidylinositol phospholipase C from Pseudomonas sp. and a PC- and PE-specific phospholipase C polypeptide.

[0431] 41. The composition according to item 40, wherein the phosphatidylinositol phospholipase C is:

[0432] a) a polypeptide comprising an amino acid sequence selected from the group consisting of:

[0433] i) amino acid residues 26-322 of SEQ ID NO: 2 or amino acid residues 1-298 of SEQ ID NO: 3;

[0434] ii) amino acid residues 26-323 of SEQ ID NO: 5 or amino acid residues 1-299 of SEQ ID NO: 6;

[0435] iii) amino acid residues 26-323 of SEQ ID NO: 8 or amino acid residues 1-299 of SEQ ID NO: 9;

[0436] iv) amino acid residues 26-323 of SEQ ID NO: 11 or amino acid residues 1-296 of SEQ ID NO: 12;

[0437] v) amino acid residues 26-322 of SEQ ID NO: 14 or amino acid residues 1-298 of SEQ ID NO: 15;

[0438] vi) amino acid residues 26-322 of SEQ ID NO: 17; and

[0439] vii) amino acid residues 28-339 of SEQ ID NO: 35; or

[0440] b) a polypeptide comprising an amino acid sequence that is at least 75% identical to one of the amino acid sequences in a); or

[0441] c) A functional fragment of a) or b).

[0442] 42. The composition according to item 41, wherein the phosphatidylinositol phospholipase C is the polypeptide of any one of items 12-20.

[0443] 43. The composition according to any one of items 38 to 42, wherein the PC and PE-specific phospholipase C polypeptides are:

[0444] a) a polypeptide comprising an amino acid sequence selected from the group consisting of:

[0445] i) amino acid residues 34-278 of SEQ ID NO: 19 or amino acid residues 1-246 of SEQ ID NO: 20;

[0446] ii) amino acid residues 25-283 of SEQ ID NO: 22 or amino acid residues 39-283 of SEQ ID NO: 22;

[0447] iii) amino acid residues 25-283 of SEQ ID NO: 24 or amino acid residues 39-283 of SEQ ID NO: 24 or amino acid residues 1-260 of SEQ ID NO: 25;

[0448] iv) amino acid residues 39-283 of SEQ ID NO:27;

[0449] v) amino acid residues 52-289 of SEQ ID NO: 29 or amino acid residues 1-263 of SEQ ID NO: 30

[0450] vi) amino acid residues 21-282 of SEQ ID NO: 32 or amino acid residues 38-282 of SEQ ID NO: 32;

[0451] vii) amino acid residues 25-280 of SEQ ID NO: 38 or amino acid residues 36-280 of SEQ ID NO: 38; and

[0452] viii) Purafine; or

[0453] b) a polypeptide comprising an amino acid sequence that is at least 75% identical to one of the amino acid sequences in a); or

[0454] c) A functional fragment of a) or b).

[0455] Examples

[0456] Strains and DNA

[0457] DNA encoding the PLC of SEQ ID NO: 2 was cloned from a Pseudomonas species isolated from a seaweed sample collected in Denmark.

[0458] The DNA encoding the PLC of SEQ ID NO: 19 was cloned from a Bacillus sp. obtained from a soil sample collected in Australia in 1990.

[0459] Codon-optimized DNA encoding the publicly known PLC was ordered from the companies Geneart (SEQ ID NO: 22, SEQ ID NO: 27 and SEQ ID NO: 29) and Gen9 (SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14).

[0460] In the following examples, the phospholipase C enzymes of the invention are referred to by SEQ ID NO. If the SEQ ID NO includes a signal peptide, it is understood that the reference is to the mature sequence of that SEQ ID NO.

[0461] Example 1: Cloning and expression

[0462] The gene encoding the phospholipase is cloned from the strain indicated above by conventional techniques, or ordered as a synthetic gene and inserted into a suitable plasmid. The gene is expressed by replacing the natural secretion signal with an additional alanine at the C-terminus with a secretion signal having the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA (SEQ ID NO:33). This results in a recombinant mature polypeptide with an alanine before the N-terminus of the mature wild-type sequence. The genes encoding SEQ ID NOs:22, 27, and 38 were cloned using the same strategy, but no additional alanine was added to the C-terminus of the signal peptide. Therefore, the recombinant mature polypeptide does not contain an alanine before the N-terminus of the mature wild-type sequence.

[0463] A clone with the correct recombinant gene sequence was selected and the corresponding plasmid was integrated into the Bacillus subtilis host cell genome (pectate lyase locus) by homologous recombination and the gene construct was expressed under the control of a triple promoter system as described in WO 99 / 43835. The gene encoding chloramphenicol acetyltransferase was used as a marker (as described in Diderichsen et al., 1993, Plasmid 30: 312-315).

[0464] The chloramphenicol resistant transformant was analyzed by PCR to verify the correct size of the amplified fragment. The recombinant bacillus subtilis clone of the expression construct comprising integration was selected and cultivated in a 500mL Erlenmeyer flask (Erlenmeyer flask) with baffles on a rotary shaker. Each Erlenmeyer flask comprised 100ml of culture medium based on yeast extract. This clone was cultivated 5 days at 30°C. The results comprised the enzyme of supernatant and the enzyme was purified as described in Example 2.

[0465] Example 2: Phospholipase C purification

[0466] Purification of the mature peptide of SEQ ID NO: 3:

[0467] use The cell-free culture buffer was exchanged to 50 mM MES pH 6.5 using a packed bed of G-25 resin. The collected fractions were loaded onto a Source 15S cation exchanger and eluted using a gradient of 0-100% 50 mM MES + 0.5 M NaCl pH 6.5 in 10 CV. The fractions were analyzed by SDS-PAGE (reducing conditions) and pooled based on purity.

[0468] Purification of the mature polypeptide of SEQ ID NO: 20:

[0469] Initially, decylamine agarose (Acetyleret Decylaminagarose, catalog number CS76, UpFront Cromatography A / S, Parkalle 42, 2100 The impurity capture step was performed using a packed bed of 10 μl (10 μl) PBS (10 μl, ... The flow-through and wash fractions were pooled and buffer exchanged to 50 mM MES pH 6.0 on a packed bed of G-25 resin. The collected fractions were loaded onto a Source 15Q anion exchanger and eluted using a gradient of 0-100% 50 mM MES + 0.5 M NaCl pH 6.0 in 10 CV. The fractions were analyzed by SDS-PAGE (reducing conditions) and pooled based on purity. The pooled fractions were concentrated by using a centrifugal filter device w / 10 kDa MWCO and loaded onto a HiLoad® equilibrated with 20 mM MES + 125 mM NaCl pH 6.0. TM 26 / 60 Superdex 200 pg gel filtration column. Fractions were analyzed by SDS-PAGE (reducing conditions) and pooled based on purity.

[0470] Example 3: Molecular weight and N-terminal sequence of PLC

[0471] Determination of molecular weight:

[0472] Use Bruker micro-TOF focusing electrospray mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) to carry out complete molecular weight analysis.These samples are diluted to 1mg / ml in MQ water.On-line washing dilution sample at MassPREP online desalting column (2.1x 10mm, part number 186002785, Waters), and be introduced into the electrospray source of 200ul / h flow velocity by Agilent LC system.Carry out data analysis (Bruker Daltonik GmbH, Bremen, Germany) with DataAnalysis 3.4 version.By deconvolution the raw data calculation molecular weight of sample in the range of 10.000 to 40.000Da.

[0473] The molecular weight of the PI-specific phospholipase of SEQ ID NO: 3 is 32.7 kDa.

[0474] The molecular weight of the PC, PE-specific phospholipase of SEQ ID NO: 20 is 27.6 kDa.

[0475] N-terminal sequencing procedures

[0476] Using Applied Biosystems Protein sequencing system was used for N-terminal sequencing analysis. The samples were purified on precast 4%-20% SDS polyacrylamide gels (Life Technologies). The gels were run according to the manufacturer's instructions and blotted to PVDF membrane (Applied Biosystems). For N-terminal amino acid sequencing, the major protein band was excised and placed on The protein sequencing system was placed in a blotting box. N-terminal sequencing was performed using a protocol for running a PVDF membrane sample (Pulse Liquid PVDF) according to the manufacturer's instructions. The N-terminal amino acid sequence was deduced from the seven chromatograms corresponding to amino acid residues 1 to 7 by comparing the retention times of the peaks in the chromatogram with the retention times of PTH amino acids in the standard chromatogram.

[0477] The N-terminal sequence of the mature polypeptide (SEQ ID NO: 3) was confirmed to be AQESPAF.

[0478] The N-terminal sequence of the mature polypeptide (SEQ ID NO: 6) was confirmed to be AQEAVGF.

[0479] The N-terminal sequence of the mature polypeptide (SEQ ID NO: 9) was confirmed to be AQEAVGF.

[0480] The N-terminal sequence of the mature polypeptide (SEQ ID NO: 20) was confirmed to be AWSADAP.

[0481] Example 4: Thermostability of Phospholipase C

[0482] The thermal stability of cellobiohydrolases was determined by differential scanning calorimetry (DSC) using a VP-capillary differential scanning calorimeter (MicroCal Inc., Piscataway, NJ, USA). The thermal denaturation temperature, Td (°C), was taken as the apex of the denaturation peak (major endothermic peak) in the thermogram (Cp vs. T) obtained after heating an enzyme solution (approximately 1 mg / ml) in buffer (50 mM sodium acetate, pH 5.5 ± 2 mM CaCl2, or 50 mM Hepes, pH 7 ± 2 mM CaCl2) at a constant programmed heating rate of 200 K / hr.

[0483] The sample solution and the reference solution (approximately 0.2 ml) were loaded into the calorimeter from storage conditions at 10° C. (reference solution: buffer without enzyme) and thermally pre-equilibrated at 20° C. for 20 minutes before a DSC scan from 20° C. to 100° C. The denaturation temperature was determined with an accuracy of approximately + / - 1° C.

[0484] Table 2: Denaturation Temperature

[0485]

[0486] Example 5: Specificity of phospholipase C for PC, PE, PI, and PA of purified PLC

[0487] use 31 P-NMR determines the substrate specificity of the phospholipase C enzyme of the present invention and Purifine. This determination follows the Figure 1 The conversion of the individual phospholipids shown in , and reveals the substrate specificity and preference of the phospholipase, and provides an indication of the optimal pH of the enzyme.

[0488] substrate

[0489] Crude soybean oil having the following specific phospholipid contents measured by P-NMR was used.

[0490] PA: 80-140ppm phosphorus (P)

[0491] PE: 140-200ppm P

[0492] PI: 70-110ppm P

[0493] PC: 130-200ppm P

[0494] In this mensuration, also used other crude oils, for example, from the crude oil of rapeseed, sunflower, corn, cottonseed, peanut, rice bran.The primary criterion is that this oil comprises minimum 30ppm every kind of specific phospholipid (significantly higher than NMR quantitative limit).Before pipetting crude oil, ensure mixing (it precipitates over time).

[0495] Buffers and enzymes

[0496] 0.2M Cs-EDTA pH 7.5 solution: EDTA (5.85 g) was dispersed in MQ-water (50 mL). The pH was adjusted to 7.5 using 50% w / w CsOH (approximately 30 mL), which completely dissolved the EDTA. MQ-water was added to a total volume of 100 mL to give a concentration of 0.2M.

[0497] Internal standard: 2 mg / mL triphenyl phosphate (TPP) solution in MeOH.

[0498] pH buffer:

[0499] 100 mM sodium acetate pH 4.0

[0500] 100 mM sodium acetate pH 5.5

[0501] 100 mM sodium acetate pH 7.0

[0502] Enzyme: Dilute to concentrations of 0.9, 0.27, and 0.09 mg enzyme protein (EP) / mL in three buffers and keep refrigerated until used on the same day.

[0503] Determination

[0504] 250 microlitres of crude oil are weighed in 2mL Eppendorf tubes, and 25 microlitre enzymes diluted in desired pH buffer are added. This produces 10, 30 and 100mg Ep / kg oil. At 50 ℃, this mixture is hatched 2h in a heated shaking table. Then add 0.500mL phosphate standard solution, 0.5mL chloroform-d (CDCl 3) and 0.5mL Cs-EDTA buffer. After 30sek vibration and subsequent centrifugation (desktop centrifuge, 3min, 13,400rpm), obtain phase separation. Lower phase is transferred to NMR-tube. With 128 scans, 5 seconds delay operation 31 P NMR. Integrate all signals. Assign values ​​(approximate ppm at 25°C): 1.7 (PA), -0.1 (PE), -0.5 (PI), -0.8 (PC). Signal positions can vary significantly depending on the exact pH, temperature, sample concentration, etc. Calculate the concentration of each species in "ppm P," i.e., mg elemental phosphorus / kg oil sample. Thus, ppm P = I / I(IS)*n(IS)*M(P) / m(oil). Calculate the % remaining phospholipids as the ratio of the phospholipid concentration in the enzyme-treated sample to the same concentration in the blank sample.

[0505] result

[0506] The results are summarized in Tables 3 to 10 below.

[0507] Table 3: Specificity of phospholipase of SEQ ID NO: 3

[0508]

[0509]

[0510] Table 4: Specificity of the phospholipase of SEQ ID NO: 20.

[0511]

[0512] Table 5: Specificity of the phospholipase of SEQ ID NO: 22 (amino acids 25-283).

[0513]

[0514] Table 6: Specificity of the phospholipase of SEQ ID NO: 27.

[0515]

[0516] Table 7: Specificity of the phospholipase of SEQ ID NO: 30.

[0517]

[0518]

[0519] Table 8: Specificity of the phospholipase of SEQ ID NO: 36.

[0520]

[0521] Table 9: Specificity of the phospholipase of SEQ ID NO: 38 (amino acids 36-280).

[0522]

[0523] For activity comparison, the performance of Purifine is shown below.

[0524] Table 10: Specificity of Purifine.

[0525]

[0526] Purifine concentration was estimated to be 15 mg / mL.

[0527] Example 6: Specificity of phospholipase C for PC, PE, PI, and PA in crude enzyme supernatant

[0528] In addition to the results for the purified enzyme samples in Example 5, the phospholipase activity of several PLC homologs was tested using crude, undiluted PLC supernatant in crude soybean oil at a ratio of 1:10 (v / v). The enzyme concentration was unknown and there was no pH control, otherwise the protocol of Example 5 was followed.

[0529] Table 11: Phospholipid hydrolysis with crude undiluted PLC containing supernatant

[0530]

[0531] Example 7: Degumming determination

[0532] The performance of the combination of the phospholipase C enzyme of the present invention and Purifine together with PI-specific and PC, PE-specific phospholipase C enzymes was tested in a degumming assay simulating industrial scale degumming. After degumming, the assay measured the following parameters in the oil phase:

[0533] a) Diacylceride content measured by high performance liquid chromatography (HPLC) coupled with an evaporative light scattering detector (ELSD), or an electrospray detector (Corona Veo).

[0534] b) Quantification of individual phospholipid species by liquid chromatography quadrupole time-of-flight mass spectrometry (LC / TOF / MS): phosphatidylcholine (PC); phosphatidylinositol (PI); phosphatidylethanolamine (PE); and phosphatidic acid (PA)

[0535] c) Total phosphorus reduction by inductively coupled plasma optical emission spectrometry (ICP-OES).

[0536] The phospholipid composition of crude soybean oil 2 or oil 3 used in the experiments is indicated in Table 12. The composition was measured by LC / MS as phosphorus derived from each phospholipid species.

[0537] Table 12: Phospholipid composition of crude oil (mg / kg phosphorus)

[0538]

[0539]

[0540] Degumming determination

[0541] Initially acid / alkali pretreatment (or not pretreatment) crude soybean oil (75g), to promote insoluble phosphatide salt conversion into more hydratable form, and ensure the environment that is applicable to this enzyme.By following completion acid / alkali pretreatment, use orthophosphoric acid to carry out acid addition based on the amount of oil equal to 0.05% (100% pure orthophosphoric acid), and in ultrasonic bath (BRANSON3510), mix 5min, and in rotator, hatch 15min, carry out alkali neutralization for the 4M NaOH that pure orthophosphoric acid equivalent (from 0.5 to 1.5) uses in ultrasonic bath subsequently and continue 5min.In 100ml centrifuge tube, cylindrical, conical bottom, in low water system (overall based on 3% water of oil amount), carry out enzyme reaction.Sample is carried out supersound process 5min, subsequently in heating drying, under selected temperature (from 50 ℃ to 60 ℃), hatch selected incubation time (from 1 to 5 hour) when stirring with 20rpm. To separate the mixture into an oil phase and a heavy water / gel phase, the samples were centrifuged at 700 g, 85° C. for 15 min (Koehler Instruments, K600X2 oil centrifuge).

[0542] a) Diacylglycerol measurement

[0543] The HPLC-ELSD or HPLC-Corona Veo method (using DIONEX equipment and Lichrocart Si-60, 5 μm, Lichrosphere 250-4 mm, Merck columns) is based on the principles of AOCS official method Cd 11d-96 and quantifies diacylglycerol content down to 0.1 wt%.

[0544] b) Quantitative analysis of phospholipids by LCMS / MS

[0545] Liquid chromatography coupled to a triple quadrupole mass spectrometer (LC / MS / MS) or to a quadrupole mass spectrometer time-of-flight (LC / TOF / MS) was used to quantify the individual phospholipid species: phosphatidylcholine (PC); phosphatidylinositol (PI); phosphatidylethanolamine (PE) and phosphatidic acid (PA). The sensitivity of the assay was down to less than 1 mg phosphorus / kg oil for PC, PE and PI (ppm), and less than 10 mg phosphorus / kg for PA. The oil sample was dissolved in chloroform. The extract was then analyzed on an LC-TOF-MS (or on an LC-MS / MS if a lower detection limit was desired) using the following setup.

[0546] LC-Setup

[0547]

[0548]

[0549] MS-Settings

[0550]

[0551] The data were processed using MassLynx version 4.1 software. In the following examples, the method is referred to as LCMS.

[0552] c) Phosphorus / phospholipid measurement

[0553] The ICP-OES quantifies phosphorus (P) content down to 4 ppm and other metals such as Ca, Mg, Zn with an accuracy of approximately ±1 ppm P.

[0554] Examples 8 to 12 below describe the results obtained using the degumming assay of this example.

[0555] Example 8: Robustness of enzyme pretreatment to different acidities of the oil

[0556] The PI-specific phospholipase C of SEQ ID NO: 3 was used in a degumming assay that tested different acid / base pretreatments of crude oil 3. The diglyceride content was measured after enzymatic degumming at 50° C. and 60° C. for 1 and 3 hr. The results are shown in Table 13.

[0557] Table 13. Increase in diacylglycerols after enzymatic treatment of acid / base pretreated soybean oil measured by HPLC-ELSD.

[0558]

[0559]

[0560] In the degumming assay, the PI-specific phospholipase C of SEQ ID NO: 3 converted PI-phospholipids into diacylglycerol within a few hours. Based on the following assumption, complete conversion was achieved after 3 hours: 182ppm P (measured by LCMS) derived from PI equaled 0.50% PI-phospholipids (Mw PI about 857g / mol, Mw P about 31g / mol) equaled 0.40% Dg increase (80% phospholipid molecules). The enzyme showed good performance in water degumming (no acid / base) as well as in acid-assisted degumming before alkali neutralization with various concentrations of NaOH. This confirms its robustness to various pH conditions in the degumming process.

[0561] Example 9: Effect of enzyme dosage

[0562] In the degumming assay, the PI-specific phospholipase C of SEQ ID NO: 3 was used in crude oil 3 at various enzyme dosages. The diglyceride content was measured after enzymatic degumming at 60° C. for 1, 2, 3, and 5 hr (oil pretreated with 0.05% phosphoric acid / 1.5 equivalents of NaOH). These results are shown in Table 14.

[0563] Table 14: Increase in diacylglycerol after enzymatic treatment of acid / base pretreated soybean oil measured by HPLC-ELSD.

[0564]

[0565] In the degumming assay, the PI-specific phospholipase C of SEQ ID NO: 3 converted PI-phospholipids into diacylglycerols in 1-5 hours at enzyme dosages ranging from 1 to 20 mg Ep / kg oil. The data demonstrated a dose-response effect with faster conversion of phospholipids to diacylglycerols at higher enzyme dosages. In all cases, the PI content was reduced to less than 1 mg / kg oil (measured by LCMS) after degumming, demonstrating that the enzyme attacks the PI species.

[0566] Example 10: Combination of PI-specific PLC and PC, PE-specific PLC at 50°C

[0567] In a degumming assay, crude oil 3 pretreated with 0.05% phosphoric acid / 1.5 equivalents of NaOH was used in a PI-specific phospholipase C of SEQ ID NO: 3 in combination with PC, PE-specific phospholipase Purifine PLC, and PLC of SEQ ID NO: 22 at 50°C. Diacylglycerol content was measured along with metal content after 1, 2, 3, and 5 hours of enzymatic degumming. The results are shown in Tables 15A+B.

[0568] Table 15A: Increase in diacylglycerol after enzymatic treatment of 0.05% phosphoric acid / 1.5 eq. NaOH pretreated soybean oil as measured by HPLC-ELSD.

[0569]

[0570] Table 15B: Ca, Mg, P content (mg / kg oil) measured by ICP-OES after 5 hours of enzyme treatment

[0571]

[0572] Degumming at 50°C with a PI-specific phospholipase C of SEQ ID NO: 3 in combination with a PC- and PE-specific PLC (purifine PLC or SEQ ID NO: 22) resulted in significant diacylglycerol formation. The combined effect was evident by a greater increase in DG compared to the individual solutions. Phosphorus content in the degummed oil was reduced to the desired final level of less than 5 mg / kg, equivalent to 'complete' conversion.

[0573] Example 11: PI-specific PLC combined with PC and PE-specific PLC at 60°C

[0574] In the degumming assay, diacylglycerol content was measured in crude oil 3 pretreated with 0.05% phosphoric acid / 1.5 equivalents of NaOH at 60° C. after enzymatic degumming using PI-specific phospholipase C of SEQ ID NO: 3, alone or in combination with PC, PE-specific PLC of SEQ ID NO: 22, for 1, 2, 3, and 5 hr. These results are shown in Table 16.

[0575] Table 16: Increase in diacylglycerol after enzymatic treatment of 0.05% phosphoric acid / 1.5 eq. NaOH pretreated crude soybean oil as measured by HPLC-ELSD.

[0576]

[0577] Compared to the effect of the individual enzymes, degumming with the PI-specific phospholipase C of SEQ ID NO: 3 in combination with the PC, PE-specific PLC of SEQ ID NO: 22 at 60°C resulted in a combined effect and converted most of the phospholipids (up to 87% under the conditions tested (60°C, 5 hours)). The calculation was based on the following assumptions: a total of 864 ppm P measured by LC / MS equals 2.15 wt% phospholipids (average Mw of about 772 g / mol), which equals a maximum achievable 1.72% Dg increase (80% of phospholipid molecules). The LCMS results showed less than 4 ppm P from PI, PC, and PE in the degummed oil sample, confirming that the blend attacks PC, PE, and PI phospholipid species.

[0578] Example 12: PI-specific PLC combined with PC and PE-specific PLC at 60°C

[0579] In a degumming assay, PI-specific phospholipase C of SEQ ID NO: 3 was used alone and in combination with Purifine PLC or SEQ ID NO: 20 in crude oil 2 pretreated with 0.05% phosphoric acid / 1.5 equivalents of NaOH at 60°C. After 3 and 5 hours of enzymatic degumming, the diacylglycerol content was measured. These results are shown in Table 17.

[0580] Table 17: Increase in diacylglycerol after enzymatic treatment of 0.05% phosphoric acid / 1.5 equivalents NaOH pretreated crude soybean oil as measured by HPLC-ELSD.

[0581]

[0582] As determined by LC / MS, in the crude oil used, the phospholipid species PI, PE and PC that were hydrolyzed by the enzyme accounted for 60% of the total 732ppm of phospholipids, which, when fully hydrolyzed, equated to a maximum DG increase of 0.86%. Compared to the effect of the individual enzymes, degumming with the PI-specific phospholipase C of SEQ ID NO: 3 in combination with PC, PE-specific PLC or Purifine PLC of SEQ ID NO: 20 resulted in a combined effect, with up to 51% of the total phospholipids converted in 5 hours at 60°C. In the crude oil sample, this corresponds to the hydrolysis of most of the PI, PE and PC. Calculations were based on an average MW of 772g / mol for the phospholipids.

[0583] Example 13: Degumming using PI-specific PLC in combination with PC and PE-specific PLC at 60°C

[0584] In a degumming assay, PC, PE-specific phospholipase C (SEQ ID NO: 22 (amino acids 25-283), SEQ ID NO: 25, and SEQ ID NO: 27) were used alone or in combination with the PI-specific phospholipase C of SEQ ID NO: 3 in crude oil 5 at 60°C. The phospholipases were dosed at 2 or 4 ml per 50 g volume of filtered broth, in addition to Purifine, which was dosed at 4 mg enzyme protein per kg of oil. The increase in diacylglycerol after 2 and 5 hours of enzymatic degumming is shown in Table 18. The water-degummed oil was post-treated with 0.09% phosphoric acid / 0.5 equivalent NaOH before measuring phosphorus content by ICP method.

[0585] Table 18: Increase in diacylglycerol measured by HPLC-Corona Veo after enzyme treatment and phosphorus content measured by ICP after oil post-treatment with 0.09% phosphoric acid / 0.5 eq. NaOH.

[0586]

[0587] As determined by LC / MS, in the crude oil used, the phospholipid species PI, PE, and PC that were hydrolyzed by the enzyme accounted for 81% of the total 583 ppm of phospholipids, equivalent to a maximum DG increase of 0.95% when fully hydrolyzed. Calculations were based on a conversion factor of 0.0025 from phosphorus to phospholipids and the fact that diacylglycerols constitute 80% of the phospholipid molecules. Degumming with the PI-specific phospholipase C of SEQ ID NO: 3 in combination with PC, PE-specific PLC of SEQ ID NO: 22 (amino acids 25-283), SEQ ID NO: 25, or SEQ ID NO: 27 resulted in a combined effect, with up to 70% of the total available phospholipids converted in 5 hours at 60°C compared to the effect of the individual enzymes.

[0588] Example 14: Degumming using PI-specific PLC in combination with PC, PE specific PLC at 60°C.

[0589] In a degumming assay, crude oil 5 was used at 60°C, and a PC, PE-specific phospholipase C of SEQ ID NO: 22 (amino acids 25-283) was used alone or in combination with PI-specific phospholipase C of SEQ ID NO: 9 and SEQ ID NO: 15. The PI-specific phospholipase C was dosed at a volume of filtered fermentation broth (2 ml) per 50 g of crude oil, while the PC, PE-specific phospholipase C was dosed at mg enzyme protein / kg oil (4 mg EP / kg oil) purified. The increase in diacylglycerol after 2 and 5 hours of enzymatic degumming is shown in Table 19. The water-degummed oil was post-treated with 0.09% phosphoric acid / 0.5 equivalents of NaOH before measuring the phosphorus content by the ICP method.

[0590] Table 19: Increase in diacylglycerol measured by HPLC-Corona Veo after enzyme treatment and phosphorus content measured by ICP after oil post-treatment with 0.09% phosphoric acid / 0.5 eq. NaOH.

[0591]

[0592] As determined by LC / MS, the phospholipid species PI, PE and PC hydrolyzed by the enzymes accounted for 81% of the total 583 ppm of phospholipids in the crude oil used, which equated to a maximum of 0.95% DG increase when fully hydrolyzed. Degumming with PC, PE-specific phospholipase C of SEQ ID NO: 22 (amino acids 25-283) in combination with PI-specific PLCs of SEQ ID NO: 9 and 15 resulted in a combined effect, with conversion of up to 70% of the total available phospholipids at 60° C. in 2 hours, compared to the action of the individual enzymes.

[0593] Example 15: Water degumming using PI-specific PLC in combination with PC, PE specific PLC at 60°C.

[0594] In a degumming assay, a PI-specific phospholipase C of SEQ ID NO: 3 was used alone or in combination with PC, PE-specific phospholipase C of SEQ ID NO: 22 (amino acids 25-283), SEQ ID NO: 25, and SEQ ID NO: 27 (amino acids 25-283) in crude oil 4 at 60°C. Purifine and SEQ ID no 22 (amino acids 25-283) are given as mg purified enzyme protein / kg oil, while the remainder is given as volume filtered broth / 40 g crude oil. The increase in diacylglycerol after 2 and 5 hours of enzymatic water degumming (no acid / base oil treatment) is shown in Table 20, along with the phosphorus content measured by ICP.

[0595] Table 20: Increase in diacylglycerol measured by HPLC-Corona Veo and phosphorus content measured by ICP after enzyme treatment.

[0596]

[0597]

[0598] Degumming with a PI-specific phospholipase C of SEQ ID NO: 3 in combination with a PC, PE-specific PLC (SEQ ID NO: 22 (amino acids 25-283), SEQ ID NO: 25, and SEQ ID NO: 27 (amino acids 25-283)) resulted in significant diacylglycerol formation. As determined by LC / MS, the phospholipid species PI, PE, and PC hydrolyzed by the enzymes accounted for 98% of the total 974 ppm of phospholipids in the crude oil used, equivalent to a maximum DG increase of 1.91% upon complete hydrolysis. Calculations were based on a conversion factor of 0.0025 from phosphorus to phospholipids, with diacylglycerols constituting 80% of phospholipid molecules. Degumming with a PI-specific phospholipase C of SEQ ID NO: 3 in combination with a PC, PE-specific PLC of SEQ ID NO: 27 resulted in a combined effect, converting up to 65% of the total available phospholipids in 5 hours at 60°C, compared to the action of the individual enzymes. The phosphorus content in the degummed oil was reduced to less than 5 mg / kg.

[0599] Example 16: Water degumming using PI-specific PLC in combination with PC and PE-specific PLC at 60°C

[0600] In degumming assays, crude oil 4 was used at 60° C., and the PC, PE-specific phospholipase C of SEQ ID NO: 22 (amino acids 25-283) was used alone or in combination with the PI-specific phospholipase C of SEQ ID NO: 9 and SEQ ID NO: 15. The PI-specific phospholipase C was dosed at 2 ml of filtered fermentation broth per 50 g of crude oil, while the PC, PE-specific phospholipase C was dosed at 4 mg enzyme protein / kg oil. The diacylglycerol increase after 2 and 5 hours of enzymatic water degumming (no acid / alkali oil treatment) and the phosphorus content measured by ICP are shown in Table 21.

[0601] Table 21: Increase in diacylglycerol measured by HPLC-Corona Veo and phosphorus content measured by ICP after enzyme treatment.

[0602]

[0603]

[0604] Degumming with a PC, PE-specific phospholipase C of SEQ ID NO: 22 (amino acids 25-283) in combination with PI-specific phospholipase C of SEQ ID NO: 9 and 15 resulted in significant diacylglycerol formation. As determined by LC / MS, the phospholipid species PI, PE, and PC hydrolyzed by the enzymes accounted for 98% of the total 974 ppm of phospholipids in the crude oil used, equivalent to a maximum of 1.91% DG increase when fully hydrolyzed. Degumming with a PC, PE-specific PLC of SEQ ID NO: 22 (amino acids 25-283) in combination with PI-specific phospholipase C of SEQ ID NO: 9 and 15 resulted in a combined effect compared to the action of the individual enzymes, with up to 69% of the total available phospholipids converted at 60° C. in 5 hours. The phosphorus content in the degummed oil was reduced to less than 5 mg / kg.

[0605] Example 17: Water degumming using PI-specific PLC in combination with PC, PE specific PLC at 60°C.

[0606] In degumming assays, crude oil 4 was used at 60° C. PC, PE-specific phospholipase C of SEQ ID NO: 22 (amino acids 25-283) was used alone or in combination with PI-specific phospholipase C of SEQ ID NO: 3 and SEQ ID NO: 6. The phospholipase was dosed at 4 ml filtered broth per 50 g crude oil. The increase in diacylglycerol after 2 and 5 hours of enzymatic water degumming (no acid / base oil treatment) is shown in Table 22, along with the phosphorus content measured by ICP after 5 hours.

[0607] Table 22: Increase in diacylglycerol measured by HPLC-Corona Veo and phosphorus content measured by ICP after enzyme treatment.

[0608]

[0609] Degumming with the PC, PE-specific phospholipase C of SEQ ID NO: 22 (amino acids 25-283) in combination with the PI-specific phospholipase C of SEQ ID NO: 3 and 6 resulted in a combined effect compared to the action of the individual enzymes, with up to 65% conversion of the total available PI, PC, PE phospholipids in 5 hours at 60° C. The phosphorus content in the degummed oil was reduced to the desired final level of less than 5 mg / kg, equivalent to 'complete' P removal / reduction.

[0610] Example 18: Degumming using PI-specific PLC (SEQ ID NO: 38, amino acids 25-280) in a degumming assay at 60°C.

[0611] In a degumming assay, a PC, PE-specific phospholipase C of SEQ ID NO: 38 (amino acids 25-280) was used at 60°C using crude oil 5 (different oils). Crude oil 5 was pretreated with 0.09% phosphoric acid and 1.5 molar equivalents of NaOH prior to incubation with the enzyme. The phospholipase was dosed at 30 mg enzyme protein / kg oil. The increase in diacylglycerol was measured by HPLC-Corona Veo after 2, 4, 6, and 24 hours of enzymatic degumming, along with the phosphorus content measured by ICP after 24 hours, are shown in Table 23.

[0612] Table 23: Increase in diacylglycerol measured by HPLC-Corona Veo and phosphorus content measured by ICP after enzyme treatment.

[0613]

[0614] As determined by LC / MS, the phospholipid species PC and PE that were hydrolyzed by the enzyme accounted for 70% of the total 583 ppm of phospholipids in the crude oil used, which equates to a maximum DG increase of 0.82% upon complete hydrolysis. Calculations were based on a conversion factor of 0.0025 from phosphorus to phospholipids and the fact that diacylglycerols constitute 80% of the phospholipid molecules. Degumming with phospholipase C of SEQ ID NO: 38 (amino acids 25-280) at 60° C. for 2 hours converted up to 53% of the total available phospholipids.

[0615] Example 19: Citric Acid / Base Pretreatment of SEQ ID NO: 22 (amino acids 39-283) and SEQ ID NO: 38 (amino acids 36-280) Tested in Degumming Assay at 60°C

[0616] In the degumming assay, crude oil 5 (different oils) was used at 60°C, using PC, PE-specific phospholipase C of SEQ ID NO: 22 (amino acids 39-283) and PC, PE-specific phospholipase C of SEQ ID NO: 38 (amino acids 36-280). Crude oil 5 was pretreated with 0.065% citric acid and 1.5 molar equivalents of NaOH prior to incubation with the enzymes. The phospholipase was dosed at 4 ml filtered broth per 50 g crude oil. The increase in diacylglycerol after 2 and 5 hours of enzymatic degumming, along with the phosphorus content measured by ICP after 2 hours, is shown in Table 24.

[0617] Table 24: Increase in diacylglycerol measured by HPLC-Corona Veo and phosphorus content measured by ICP after enzyme treatment.

[0618]

[0619]

[0620] The invention described and claimed herein is not limited to the scope of the particular aspects disclosed herein, as these aspects are intended to serve as illustrations of several aspects of the invention. It is contemplated that any equivalent aspects are within the scope of the invention. Indeed, various modifications of the invention, other than those shown and described herein, will become clear to those of ordinary skill in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the event of a conflict, the present disclosure, including definitions, will prevail.

Claims

1. A method for reducing the phospholipid content in an oil composition, the method comprising a) providing an oil composition comprising a certain amount of phospholipids, b) contacting the oil composition with a phosphatidylinositol phospholipase C and PC and PE-specific phospholipase C under conditions sufficient for the enzymes to react with the phospholipids to produce diacylglycerol and phosphate; and, c) separating the phosphate ester from the oil composition, in, The phosphatidylinositol phospholipase C is a polypeptide consisting of SEQ ID NO: 2 or amino acids 26 to 322 of SEQ ID NO: 2 or amino acids 1 to 298 of SEQ ID NO:

3.

2. The method of claim 1, wherein the oil is an edible oil.

3. The method of claim 1, wherein the oil is selected from the group consisting of crude oil, water degummed oil, caustic refined oil and acid degummed oil.

4. The method of claim 1, wherein the oil comprises phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol.

5. The method of claim 4, wherein the oil comprises at least 50 ppm phosphorus derived from phosphatidylinositol.

6. The method according to any one of claims 1 to 5, wherein the PC and PE-specific phospholipase C are a) a polypeptide comprising an amino acid sequence selected from the group consisting of: i) amino acid residues 34-278 of SEQ ID NO: 19 or amino acid residues 1-246 of SEQ ID NO: 20; vii) amino acid residues 25-283 of SEQ ID NO: 22 or amino acid residues 39-283 of SEQ ID NO: 22; viii) amino acid residues 25-283 of SEQ ID NO: 24 or amino acid residues 39-283 of SEQ ID NO: 24 or amino acid residues 1-260 of SEQ ID NO: 25; iv) amino acid residues 25-283 of SEQ ID NO:27 or amino acid residues 39-283 of SEQ ID NO:27; v) amino acid residues 28-289 of SEQ ID NO:29 or amino acid residues 52-289 of SEQ ID NO:29 or amino acid residues 1-263 of SEQ ID NO:30; vi) amino acid residues 21-282 of SEQ ID NO: 32 or amino acid residues 38-282 of SEQ ID NO: 32; vii) amino acid residues 25-280 of SEQ ID NO: 38 or amino acid residues 36-280 of SEQ ID NO: 38; and viii) Purifine; or b) A polypeptide comprising an amino acid sequence that is at least 95% identical to one of the amino acid sequences in a).

7. The method according to any one of the preceding claims 1 to 5, wherein the PC and PE-specific phospholipase C is a polypeptide having a length of 220-280 amino acid residues.

8. The method according to any one of the preceding claims 1 to 5, wherein the oil is contacted with: 0.5-200 mg enzyme protein / Kg oil of the phosphatidylinositol phospholipase C and 0.5-200 mg enzyme protein / Kg oil of the PC and PE-specific phospholipase C.

9. A polypeptide having phosphatidylinositol phospholipase C activity, the polypeptide consisting of SEQ ID NO: 2 or amino acids 26 to 322 of SEQ ID NO: 2 or amino acids 1 to 298 of SEQ ID NO:

3.

10. The polypeptide of claim 9, having a thermal denaturation temperature of at least 60°C as determined by differential scanning calorimetry.

11. The polypeptide of claim 10, wherein the denaturation temperature is determined as the apex of the denaturation peak in a thermogram obtained after heating a 1 mg / ml solution of the polypeptide in 50 mM sodium acetate, pH 5.5 buffer, or 50 mM Hepes, pH 7 buffer at a constant programmed heating rate of 200 K / hr.

12. The polypeptide of claim 9, wherein the polypeptide is capable of reducing the phosphatidylinositol content of crude soybean oil by 50% or more, the reduction in phosphatidylinositol content being achieved by adding 100 mg enzyme protein / kg oil and incubating the oil and enzyme at 50° C. at pH 5.5 for 2 hours. 31 Confirmed by P-NMR.

13. The polypeptide of claim 9, which is capable of reducing the phosphorus content of crude soybean oil to 20 mg / kg oil or less as measured by inductively coupled plasma optical emission spectrometry after incubation of 4 mg enzyme protein / kg oil in a low-water system comprising 3% water based on the amount of oil at 50°C-60°C for 5 hours.

14. The polypeptide of claim 13, wherein the crude soybean oil comprises 80-140 ppm phosphorus present as phosphatidic acid, 140-200 ppm phosphorus present as phosphatidylethanolamine, 70-110 ppm phosphorus present as phosphatidic acid, and 130-200 ppm phosphorus present as phosphatidylcholine; the phosphorus content being determined by 31 P-NMR measurement.

15. The polypeptide according to claim 13, wherein the reduction in phosphorus content is obtained in an oil degumming process comprising the steps of: i) treating crude soybean oil with acid / base by adding 85% orthophosphoric acid solution in an amount corresponding to 0.05% of 100% pure orthophosphoric acid based on the amount of oil, mixing in an ultrasonic bath for 5 minutes, followed by incubation in a rotator for 15 minutes and neutralization with 4M NaOH base in an amount equivalent to pure orthophosphoric acid in an ultrasonic bath for 5 minutes; ii) adding the polypeptide to the oil in an amount of 4 mg enzyme protein / kg oil in a low-water system including 3% water based on the amount of oil, and subjecting the oil and the polypeptide to ultrasonic treatment for 5 minutes; iii) incubating the polypeptide and oil at 50° C.-60° C. for 5 hours with stirring at 20 rpm; iv) Centrifuge the oil and polypeptide at 700 g at 85°C for 15 minutes.

16. A polynucleotide encoding the polypeptide according to any one of claims 9 to 15.

17. A nucleic acid construct or expression vector comprising the polynucleotide of claim 16 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

18. A recombinant host cell comprising the polynucleotide of claim 16 operably linked to one or more control sequences that direct the production of the polypeptide.

19. A method for producing the polypeptide according to any one of claims 9 to 15, the method comprising: A cell is cultured under conditions conducive for production of the polypeptide, which cell, in its wild-type form, produces the polypeptide.

20. A method for producing the polypeptide of any one of claims 9 to 15, the method comprising: The host cell of claim 18 is cultured under conditions conducive for production of the polypeptide.

21. The method of claim 19 or 20, further comprising recovering the polypeptide.

22. A composition comprising the polypeptide of any one of claims 9-15.

23. A composition comprising a mixture of a phosphatidylinositol phospholipase C from Pseudomonas and a PC and PE-specific phospholipase C polypeptide, wherein: The phosphatidylinositol phospholipase C is a polypeptide consisting of SEQ ID NO: 2 or amino acids 26 to 322 of SEQ ID NO: 2 or amino acids 1 to 298 of SEQ ID NO:

3.

24. The composition of claim 23, wherein the PC and PE-specific phospholipase C polypeptide is: a) a polypeptide comprising an amino acid sequence selected from the group consisting of: i) amino acid residues 34-278 of SEQ ID NO: 19 or amino acid residues 1-246 of SEQ ID NO: 20; ii) amino acid residues 25-283 of SEQ ID NO: 22 or amino acid residues 39-283 of SEQ ID NO: 22; iii) amino acid residues 25-283 of SEQ ID NO: 24 or amino acid residues 39-283 of SEQ ID NO: 24 or amino acid residues 1-260 of SEQ ID NO: 25; iv) amino acid residues 39-283 of SEQ ID NO:27; v) amino acid residues 52-289 of SEQ ID NO: 29 or amino acid residues 1-263 of SEQ ID NO: 30 vi) amino acid residues 21-282 of SEQ ID NO: 32 or amino acid residues 38-282 of SEQ ID NO: 32; vii) amino acid residues 25-280 of SEQ ID NO: 38 or amino acid residues 36-280 of SEQ ID NO: 38; and viii) Purafine; or b) A polypeptide comprising an amino acid sequence that is at least 95% identical to one of the amino acid sequences in a).

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