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Phospholipases, Nucleic Acids Encoding Them and Methods for Making and Using Them

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Phospholipases, Nucleic Acids Encoding Them and Methods for Making and Using Them (19) TZZ¥_Z_ _T (11) EP 3 190 182 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 12.07.2017 Bulletin 2017/28 C12N 9/20 (2006.01) C12N 1/20 (2006.01) C12N 15/00 (2006.01) C07H 21/04 (2006.01) (21) Application number: 16184319.8 (22) Date of filing: 08.03.2005 (84) Designated Contracting States: • FIELDING, Roderick AT BE BG CH CY CZ DE DK EE ES FI FR GB GR San Diego, CA 92109 (US) HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR • BROWN, Robert C. San Diego, CA 92130 (US) (30) Priority: 08.03.2004 US 796907 • VASAVADA, Amit Poway, CA 92064 (US) (62) Document number(s) of the earlier application(s) in • TAN, Xuqiu accordance with Art. 76 EPC: San Diego, CA 92130 (US) 05727242.9 / 1 748 954 • BADILLO, Adrian Poway, CA 92064 (US) (27) Previously filed application: • VAN HOEK, Wilhelmus P. 08.03.2005 PCT/US2005/007908 San Diego, CA 92126 (US) • JANSSEN, Giselle (71) Applicant: DSM IP Assets B.V. San Diego, CA 92121 (US) 6411 TE Heerlen (NL) • ISAAC, Charles Carlsbad, CA 92008 (US) (72) Inventors: • BURK, Mark J. • GRAMATIKOVA, Svetlana San Diego, CA 92130 (US) San Diego, CA 92122 (US) • HAZLEWOOD, Geoff (74) Representative: Cazemier, Anne Engeline et al San Diego, CA 92130 (US) DSM Intellectual Property •LAM,David P.O. Box 4 Encinitas, CA 92024 (US) 6100 AA Echt (NL) • BARTON, Nelson R. San Diego, CA 92131 (US) Remarks: • STURGIS, Blake G. •A request for correction of the description has been Solana Beach, CA 92075 (US) filed pursuant to Rule 139 EPC. A decision on the • ROBERTSON, Dan E. request will be taken during the proceedings before San Diego, CA 92130 (US) the Examining Division (Guidelines for Examination • LI, Jincai in the EPO, A-V, 3.). San Diego, CA 92115 (US) •This application was filed on 16.08.2016 as a • KREPS, Joel A. divisional application to the application mentioned Encinitas, CA 92024 (US) under INID code 62. (54) PHOSPHOLIPASES, NUCLEIC ACIDS ENCODING THEM AND METHODS FOR MAKING AND USING THEM (57) The invention provides novel polypeptides having phospholipase activity, including, e.g., phospholipase A, B, C and D activity, patatin activity, phosphatidic acid phosphatases (PAP)) and/or lipid acyl hydrolase (LAH) activity, nucleic acids encoding them and antibodies that bind to them. Industrial methods, e.g., oil degumming, and products comprising use of these phospholipases are also provided. EP 3 190 182 A1 Printed by Jouve, 75001 PARIS (FR) EP 3 190 182 A1 Description FIELD OF THE INVENTION 5 [0001] This invention relates generally to phospholipase enzymes, polynucleotides encoding the enzymes, methods of making and using these polynucleotides and polypeptides. In particular, the invention provides novel polypeptides having phospholipase activity, nucleic acids encoding them and antibodies that bind to them. Industrial methods and products comprising use of these phospholipases are also provided. 10 BACKGROUND [0002] Phospholipases are enzymes that hydrolyze the ester bonds of phospholipids. Corresponding to their impor- tance in the metabolism of phospholipids, these enzymes are widespread among prokaryotes and eukaryotes. The phospholipases affect the metabolism, construction and reorganization of biological membranes and are involved in 15 signal cascades. 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 free fatty acid and 1-lyso-2-acylphospholipid. Phospholipase A2 (PLA2) removes the 2-position fatty acid to produce free fatty acid and 1-acyl-2-lysophospholipid. PLA1 and PLA2 enzymes can be intra- or extra-cellular, membrane-bound or soluble. Intracellular PLA2 is found in almost every mammalian cell. Phospholipase C (PLC) removes the phosphate 20 moiety to produce 1,2 diacylglycerol and phosphate ester. Phospholipase D (PLD) produces 1,2-diacylglycerophosphate and base group. PLC and PLD are important in cell function and signaling. PLD had been the dominant phospholipase in biocatalysis (see, e.g., Godfrey, T. and West S. (1996) Industrial enzymology, 299-300, Stockton Press, New York). Patatins are another type of phospholipase, thought to work as a PLA (see for example, Hirschberg HJ, et al., (2001), Eur J Biochem 268(19):5037-44). 25 [0003] Common oilseeds, such as soybeans, rapeseed, sunflower seeds, rice bran oil, sesame and peanuts are used as sources of oils and feedstock. In the oil extraction process, the seeds are mechanically and thermally treated. The oil is separated and divided from the meal by a solvent. Using distillation, the solvent is then separated from the oil and recovered. The oil is "degummed" and refined. The solvent content in the meal can be evaporated by thermal treatment in a "desolventizer toaster," followed by meal drying and cooling. After a solvent had been separated by distillation, the 30 produced raw oil is processed into edible oil, using special degumming procedures and physical refining. It can also be utilized as feedstock for the production of fatty acids and methyl ester. The meal can be used for animal rations. [0004] Degumming is the first step in vegetable oil refining and it is designed to remove contaminating phosphatides that are extracted with the oil but interfere with the subsequent oil processing. These phosphatides are soluble in the vegetable oil only in an anhydrous form and can be precipitated and removed if they are simply hydrated. Hydration is 35 usually accomplished by mixing a small proportion of water continuously with substantially dry oil. Typically, the amount of water is 75% of the phosphatides content, which is typically 1 to 1.5 %. The temperature is not highly critical, although separation of the hydrated gums is better when the viscosity of the oil is reduced at 50°C to 80°C. [0005] Many methods for oil degumming are currently used. The process of oil degumming can be enzymatically assisted by using phospholipase enzymes. Phospholipases A1 and A2 have been used for oil degumming in various 40 commercial processes, e.g., "ENZYMAX™ degumming" (Lurgi Life Science Technologies GmbH, Germany). Phosphol- ipase C (PLC) also has been considered for oil degumming because the phosphate moiety generated by its action on phospholipids is very water soluble and easy to remove and the diglyceride would stay with the oil and reduce losses; see e.g., Godfrey, T. and West S. (1996) Industrial Enzymology, pp.299-300, Stockton Press, New York; Dahlke (1998) "An enzymatic process for the physical refining of seed oils," Chem. Eng. Technol. 21:278-281; Clausen (2001) "Enzy- 45 matic oil degumming by a novel microbial phospholipase," Eur. J. Lipid Sci. Technol. 103:333-340. [0006] High phosphatide oils such as soy, canola and sunflower are processed differently than other oils such as palm. Unlike the steam or "physical refining" process for low phosphatide oils, these high phosphorus oils require special chemical and mechanical treatments to remove the phosphorus-containing phospholipids. These oils are typically refined chemically in a process that entails neutralizing the free fatty acids to form soap and an insoluble gum fraction. The 50 neutralization process is highly effective in removing free fatty acids and phospholipids but this process also results in significant yield losses and sacrifices in quality. In some cases, the high phosphatide crude oil is degummed in a step preceding caustic neutralization. This is the case for soy oil utilized for lecithin wherein the oil is first water or acid degummed. [0007] Phytosterols (plant sterols) are members of the "triterpene" family of natural products, which includes more 55 than 100 different phytosterols and more than 4000 other types of triterpenes. In general, phytosterols are thought to stabilize plant membranes, with an increase in the sterol/phospholipid ration leading to membrane rigidification. Chem- ically, phytosterols closely resemble cholesterol in structure and are thought to regulate membrane fluidity in plant membranes, as does cholesterol in animal membranes. The major phytosterols are β-sitosterol, campesterol and stig- 2 EP 3 190 182 A1 masterol. Others include stigmastanol (β-sitostanol), sitostanol, desmosterol, dihydrobrassicasterol, chalinasterol, por- iferasterol, clionasterol and brassicasterol. [0008] Plant sterols are important agricultural products for health and nutritional industries. They are useful emulsifiers for cosmetic manufacturers and supply the majority of steroidal intermediates and precursors for the production of 5 hormone pharmaceuticals. The saturated analogs of phytosterols and their esters have been suggested as effective cholesterol-lowering agents with cardiologic health benefits. Plant sterols reduce serum cholesterol levels by inhibiting cholesterol absorption in the intestinal lumen and have immunomodulating properties at extremely low concentrations, including enhanced cellular response of T lymphocytes and cytotoxic ability of natural killer cells against a cancer cell line. In addition, their therapeutic effect has been demonstrated in clinical studies for treatment of pulmonary tuberculosis, 10 rheumatoid arthritis, management of HIV-infested patients and inhibition of immune stress in marathon runners. [0009] Plant sterol esters, also referred to as phytosterol esters, were approved as GRAS (Generally Recognized As Safe) by the US Food and Drug Administration (FDA) for use in margarines and spreads in 1999. In September 2000, the FDA also issued an interim rule that allows health-claims labeling of foods containing phytosterol ester. Consequently enrichment of foods with phytosterol esters is highly desired for consumer acceptance. 15 [0010] Soybean oil is widely used and is an important foodstuff, accounting for ∼30% of the oil production from seeds and fruits. Soybeans contain only 20% oil, and the extraction is usually done by using a solvent such as hexane on a commercial scale. The recognized quality of its oil and the nutritive value of the meal protein make soya bean a primary oilseed.
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