Improved process of degumming

Abstract

The present invention relates to a process for reducing phosphorous content in oil feedstock, said process comprising steps of: a. providing an oil feedstock; b. treating the oil feedstock with an acid; c. contacting the reaction mixture of step b) in presence of one or more phospholipases under condition allowing enzymatic hydrolysis of phospholipids in the oil feedstock; and d. separation of reaction mixture of step c), wherein the acid treatment and the enzymatic hydrolysis is performed in the same tank / vessel.

Description

[0001] IMPROVED PROCESS OF DEGUMMING

[0002] REFERENCE TO A SEQUENCE LISTING

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a process for reducing phosphorous content in oil using acid and one or more enzymes having phospholipase activity.

[0006] Description of the related art

[0007] Enzymes having phospholipase degrading activity may be applied for degumming of vegetable oils to provide refined storage stable vegetable oils of neutral taste and light color suitable for consumption. The degumming process comprises removing the phospholipid compounds also known as phosphatides or “the gum” from the triglyceride rich oil fraction.

[0008] Whether intended for human consumption or as feedstock in production of oleo chemicals or biodiesel, vegetable oil needs to be pretreated to remove impurities, such as phospholipids (“gums”) and free fatty acids. The pretreatment includes Degumming, Refining (also referred to a “Neutralization”), Bleaching and Deodorization.

[0009] The purpose of the degumming process is to remove hydratable and nonhydratable phospholipids or gums present in the oil. Traditionally, the degumming process has been based on use of water extraction (“water degumming”), which involves treating the oil with water and separation of the hydratable phospholipids or gums from the triglyceride oil. Depending on the source of oil, water degumming may be combined with “acid degumming” in which the oil is treated with acid the non-hydratable gums are separated from the triglyceride oil.

[0010] Enzymatic degumming is performed on oils which have been water degummed as well as on crude oils. In the enzymatic degumming process, the phospholipids are hydrolysed in a reaction catalyzed by enzymes having phospholipase activity and are thereby converted into water soluble and water extractable components.

[0011] There are two general types of refining: “Chemical refining” (also referred to as “Alkali refining”) and “Physical refining”. Chemical refining, which comprises treatment of the oil with an alkali solution or other refining solution, is performed to reduce the free fatty acid content and will also remove other impurities such as phospholipids, proteinaceous and mucilaginous substances and color compounds. This process results in a large reduction of free fatty acids through their conversion into high specific gravity soaps, which are removed by centrifugation with some loss of neutral oil. Most phosphatides and mucilaginous substances are soluble in the oil only in an anhydrous form and upon hydration with the caustic or other refining solution are readily separated. After alkali refining, the fat or oil is water-washed to remove residual soap.

[0012] Traditionally, the degumming process has been based on water extraction, with acidic or caustic treatment followed by a separation process. Due to the emulsifying properties of the phosphatides, the degumming procedure has resulted in a loss of oil i.e., of triglycerides. However, lately enzymatic degumming has become more widespread. Enzymatic degumming is performed on oils which have been water degummed as well as crude oils. In water degumming of edible oils, a part of the phosphatides is left in the oil. That part is described by the generic term “non-hydratable phosphatides” (NHP). In the production of oils, it is essential to remove the NHP content (US 5264367). In the enzymatic degumming process, the NHP are converted by the use of phospholipase into water soluble and water extractable components.

[0013] Therefore, with the goal to maximize profit margin, it is necessary to decrease processing time, energy and chemicals consumption, oil loss and to reduce as much as possible waste production during degumming. The prior art methods do not offer the most efficient method in the shortest time possible. Therefore, a need exists for the improved process for the oil degumming with a minimal residence time and energy cost that produces degummed oils with low levels of residual phospholipids and FFA. The present invention satisfies these needs and provides other related advantages.

[0014] SUMMARY OF THE INVENTION

[0015] The present invention relates to a process for reducing phosphorous content in oil feedstock, said process comprising steps of: a. providing an oil feedstock; b. treating the oil feedstock with an acid; c. contacting the reaction mixture of step b) in presence of one or more phospholipases under condition allowing enzymatic hydrolysis of phospholipids in the oil feedstock; and d. separation of reaction mixture of step c), wherein the acid treatment and the enzymatic hydrolysis is performed in the same tank / vessel.

[0016] SEQUENCE OVERVIEW

[0017] SEQ ID NO: 1 is a phospholipase obtained from Talaromyces leycettanus.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings set forth herein are illustrative of embodiments as provided herein and are not meant to limit the scope of the invention as encompassed by the claims. Figure 1 illustrates processes for degumming oil.

[0020] DEFINITIONS

[0021] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0022] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a step" includes reference to one or more of such steps.

[0023] As used herein, "substantial" when used in reference to a quantity or amount of a material, or a specific characteristic thereof, refers to an amount that is sufficient to provide an effect that the material or characteristic was intended to provide. The exact degree of deviation allowable may in some cases depend on the specific context. Similarly, "substantially free of" or the like refers to the lack of an identified element or agent in a composition. Particularly, elements that are identified as being "substantially free of" are either completely absent from the composition or are included only in amounts which are small enough so as to have no deleterious effect on the composition.

[0024] Reference to “about” a value or parameter herein includes embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes the embodiment “X”. When used in combination with measured values, “about” includes a range that encompasses at least the uncertainty associated with the method of measuring the particular value and can include a range of plus or minus two standard deviations around the stated value. Likewise, reference to a gene or polypeptide that is “derived from” another gene or polypeptide X, includes the gene or polypeptide X.

[0025] It is understood that the embodiments described herein include “consisting” and / or “consisting essentially of” embodiments. As used herein, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments.

[0026] Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a weight range of about 1 percent to about 20 percent should be interpreted to include not only the explicitly recited concentration limits of 1 percent to about 20 percent, but also to include individual concentrations such as 2 percent, 3 percent, 4 percent, and sub-ranges such as 5 percent to 15 percent, 10 percent to 20 percent, etc.

[0027] Alkali: In the present context “alkali” refers interchangeably to a base that is soluble in water and forms hydroxide ions, such as NaOH, KOH, sodium carbonate, Ca(OH)2,and Mg(OH)2and to the solution of a base in water.

[0028] Alcoholysis: The term "alcoholysis" as used herein, refers to the reaction of an ester with a monohydric alcohol, such as ethanol, butanol, or polyhydric alcohol as glycerol, to produce an ester with a different alkyl group.

[0029] Alpha-amylase: The term “alpha amylase” means an 1 ,4-alpha-D-glucan glucanohydrolase, EC. 3.2.1.1 , which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.

[0030] Bleaching: The term “bleaching” refers to the process for removing color producing substances and for further purifying the fat or oil. Normally, bleaching is accomplished after the oil has been refined.

[0031] Cellobiohydrolase: The term “cellobiohydrolase” means a 1 ,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176) that catalyzes the hydrolysis of 1 ,4- beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1 ,4-linked glucose containing polymer, releasing cellobiose from the reducing end (cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of the chain (Teeri, 1997, Trends in Biotechnology 15: 160-167; Teeri et al., 1998, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity can be determined according to the procedures described by Lever et al., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581.

[0032] Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or “cellulase” means one or more (e.g., several) enzymes that hydrolyze a cellulosic-containing material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. The two basic approaches for measuring cellulolytic enzyme activity include: (1) measuring the total cellulolytic enzyme activity, and (2) measuring the individual cellulolytic enzyme activities (endoglucanases, cellobiohydrolases, and betaglucosidases) as reviewed in Zhang et al., 2006, Biotechnology Advances 24: 452-481. Total cellulolytic enzyme activity can be measured using insoluble substrates, including Whatman N°1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common total cellulolytic activity assay is the filter paper assay using Whatman N°1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Pure Appl. Chem. 59: 257-68).

[0033] Cellulolytic enzyme activity can be determined by measuring the increase in production / release of sugars during hydrolysis of a cellulosic-containing material by cellulolytic enzyme(s) under the following conditions: 1-50 mg of cellulolytic enzyme protein / g of cellulose in pretreated corn stover (PCS) (or other pretreated cellulosic- containing material) for 3-7 days at a suitable temperature such as 40°C-80°C, e.g., 50°C, 55°C, 60°C, 65°C, or 70°C, and a suitable pH such as 4-9, e.g., 5.0, 5.5, 6.0, 6.5, or 7.0, compared to a control hydrolysis without addition of cellulolytic enzyme protein. Typical conditions are 1 mL reactions, washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodium acetate pH 5, 1 mM MnSC>4, 50°C, 55°C, or60°C, 72 hours, sugar analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

[0034] Chemical refining: In the present application, the term “chemical refining” is used synonymously with “alkali refining” and “alkaline refining”; the term also covering “caustic refining” and ’’caustic neutralization”. Conditions facilitating hydrolysis of phospholipids: Selecting the conditions which will facilitate hydrolysis of phospholipids by one or more phospolipases is within the skill of a person skilled in the art, and includes for example adjusting pH, and / or temperature at which phospholipid degrading enzyme are active.

[0035] Crude oil: The term “crude oil” refers to (also called a non-degummed oil) a pressed or extracted oil or a mixture thereof from, e.g. vegetable or animal 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, coriander oil, corn oil, cottonseed oil, crambe oil, flax seed oil, grape seed oil, hazelnut oil, hempseed oil, jatropha oil, jojoba oil, linseed oil, macadamia nut oil, mango kernel oil, meadowfoam oil, mustard oil, neat's foot oil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil, pecan oil, pine nut oil, pistachio oil, poppy seed oil, rapeseed oil, rice bran oil, safflower oil, sasanqua oil, sesame oil, shea butter, soybean oil, sunflower seed oil, tall oil, tsubaki oil walnut oil, varieties of "natural" oils having altered fatty acid compositions via Genetically Modified Organisms (GMO) or traditional "breading" such as high oleic, low linolenic, or low saturated oils ( high oleic canola oil, low linolenic soybean oil or high stearic sunflower oils). Animal fat oil includes but not limited to cooking oil, yellow grease, tallow oil or choice white grease, poultry fat, fish oil or a blend thereof.

[0036] Degummed oil: The term "degummed oil" refers to an oil obtained after removal of non-hydratable phospholipids, hydratable phospholipids, and lecithins (known collectively as "gums") from the oil to produce a degummed oil or fat product that can be used for food production and / or non-food applications, e.g. biodiesel.

[0037] Deodorization: “Deodorization” is a vacuum steam distillation process for the purpose of removing trace constituents that give rise to undesirable flavors, colors and odors in fats and oils. Normally this process is accomplished after refining and bleaching.

[0038] Endoglucanase: The term “endoglucanase” means a 4-(1 ,3;1 ,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1 ,3-1 ,4 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components. Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481). Endoglucanase activity can also be determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40°C.

[0039] Esterification: The term "esterification" as used herein, refers to a reaction for combining an organic acid such as a fatty acid with any alcohol or polyol such as a glycerol.

[0040] Filtration: The phospholipase of the invention can be used to improve the filterability of an aqueous solution or slurry of carbohydrate origin by treating it with the phospholipase. This is particularly applicable to a solution of slurry containing a starch hydrolyzate, especially a wheat starch hydrolyzate, since this tends to be difficult to filter and to give cloudy filtrates. The treatment can be done in analogy with EP 219,269 (CPC International).

[0041] Fractionation: Fractionation is the process of separating the triglycerides in fats and oils by difference in melt points, solubility or volatility. It is most commonly used to separate fats that are solid at room temperature but is also used to separate triglycerides found in liquid oils.

[0042] Fatty acid alkyl esters (FAAE): Fatty acid alkyl esters are esters with a long carbon chain and an alkyl group, derived by transesterification fats with an alcohol. If the alcohol is methanol, the alkyl group in the fatty acid alkyl ester will be methyl, if the alcohol is ethanol, the alkyl group will be ethyl and so on.

[0043] Fatty acid feedstock: The term "fatty acid feedstock" or “vegetable oil feedstock” is defined herein as a substrate comprising triglyceride. In addition to triglyceride, the substrate may comprise diglyceride, monoglyceride, free fatty acid or any combination thereof. Any oils and fats of vegetable origin comprising fatty acids may be used as substrate for producing fatty acid alkyl esters in the process of the invention. The fatty acid feedstock may be oil selected from the group consisting of: algae oil, castor oil, coconut oil (copra oil), corn oil, cottonseed oil, flax oil, grape seed oil, hemp oil, jatropha oil, jojoba oil, mustard oil, canola oil, palm oil, palm stearin, palm olein, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, soybean oil, sunflower oil, tall oil, and oil from halophytes, or any combination thereof. The fatty acid feedstock may be crude, refined, bleached, deodorized, degummed, or any combination thereof.

[0044] Fatty acid methyl esters (FAME): Fatty acid methyl esters are esters with a long carbon chain and a methyl group, derived by transesterification of fats with methanol.

[0045] Free fatty acids (FFA): A free fatty acid is a carboxylic acid with a long carbon chain. Most naturally occurring fatty acids have an unbranched chain of an even number of carbon atoms, from 4 to 28. Free fatty acids are usually derived from fats (triglycerides (TAG), diglycerides (DAG), monoglyceride(DAG)), phospholipids or lyso-phospholipids. Triglycerides are formed by combining glycerol with three fatty acid molecules. The hydroxyl (HO-) group of glycerol and the carboxyl (-COOH) group of the fatty acid join to form an ester. The glycerol molecule has three hydroxyl (HO-) groups. Each fatty acid has a carboxyl group (-COOH). Diglycerides are formed by combining glycerol with two fatty acid molecules. Monoglycerides are formed by combining glycerol with one fatty acid molecule.

[0046] Gum: In the context of the present invention “gum”, “gums” or “gum fraction” refers to a fraction enriched in phosphatides, which is separated from the bulk of vegetable oil during a degumming process. “Gums” consist mainly of phosphatides but also contain entrained oil, contain nitrogen and sugar and meal particles.

[0047] Hydrotreated Vegetable Oil (HVO): HVO is hydrotreated vegetable oil which is a process catalyzed by inorganic heterogenic catalyst at high temperature and pressure and reacting hydrogen with the oil components to produce alkanes.

[0048] Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolytic enzyme” or “hemicellulase” means one or more (e.g., several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom and Shoham, 2003, Current Opinion In Microbiology 6(3): 219-228). Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. The substrates for these enzymes, hemicelluloses, are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups. These catalytic modules, based on homology of their primary sequence, can be assigned into GH and CE families. Some families, with an overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A). A most informative and updated classification of these and other carbohydrate active enzymes is available in the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitable temperature such as 40°C-80°C, e.g., 50°C, 55°C, 60°C, 65°C, or 70°C, and a suitable pH such as 4-9, e.g., 5.0, 5.5, 6.0, 6.5, or 7.0.

[0049] Lipid: The term "lipid" refers to phospholipids and their derivatives, triglycerides and derivatives, sterols, stands, cholesterol, sphingolipids, ceramides, fatty acids, fatty alcohols, glycolipids, proteolipids, lipopolysaccharides, ether-lipids, polar and non-polar lipids and derivatives thereof.

[0050] Lipase activity: The term “lipase activity” is defined herein as a lipolytic activity which hydrolyses the carboxylic ester bond in glyceryl tributyrate, olein, pNP-butyrate and pNP-palmitate (triacylglycerol lipase, EC 3.1.1.3).

[0051] Lysophospholipase: A “lysophospholipase” (EC 3.1.1.5) is an enzyme that can hydrolyze 2-lysophospholids to release fatty acid. Lysophospholipase activity (LLU) may be measured using egg yolk L-a-lysolecithin as the substrate with a NEFA C assay kit. 20 pl of sample is mixed with 100 pl of 20 mM sodium acetate buffer (pH 4.5) and 100 pl of 1% L- a -lysolecithin solution, and incubated at 55°C for 20 min. After 20 min, the reaction mixture is transferred to the tube containing 30 pl of Solution A in NEFA kit preheated at 37°C. After 10 min incubation at 37°C, 600 pl of Solution B in NEFA kit is added to the reaction mixture and incubated at 37°C for 10 min. Activity is measured at 555 nm on a spectrophotometer. One unit of lysophospholipase activity (1 LLU) is defined as the amount of enzyme that can increase the A550 of 0.01 per minute at 55°C.

[0052] Phospholipase: The term “phospholipase” means an enzyme that catalyzes the conversion of phospholipids into fatty acids and other lipophilic substances. Phospholipases include but not limited to such as phospholipase A, B, C, D, lysophospholipases.

[0053] Phospholipase A: In the context of the present invention the term “phospholipase A” or “phospholipase A activity” comprises enzymes having phospholipase A1 and / or phospholipase A2 activity (A1 or A2, EC 3.1.1.32 or EC 3.1.1.4), i.e., hydrolytic activity towards one or both carboxylic ester bonds in phospholipids such as lecithin. A phospholipases having both A1 and A2 activity is also referred to as a phospholipase B.

[0054] Phospholipase C: The term “phospholipase C” or “PLC activity” relates to an enzyme that removes the phosphate ester moiety from a phospholipid to produce a 1,2 diacylglycerol. Most PLC enzymes belong to the family of hydrolases and phosphodiesterases and are generally classified as EC 3.1.4.3, E.C. 3.1.4.11 or EC 4.6.1.13. Phospholipase C is specified towards one or more phospholipids, with the four most important once being phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) and phosphatidyl inositol (PI).

[0055] PC and PE-specific phospholipase C: The terms “PC and PE-specific phospholipase C” and “phospholipase C having specificity for phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE)” and “polypeptide having activity towards phosphatidylcholine (PC) and phosphatidylethanolamine (PE)” are used interchangeably. They relate to a polypeptide having activity towards phosphatidylcholine (PC), phosphatidylethanolamine (PE). In addition to the PC and PE specificity it may also have some activity towards phosphatidic acid (PA) and phosphatidyl inositol (PI).

[0056] Pl-Specific Phospholipase C: The terms “Pl-specific phospholipase C”, “Phosphatidylinositol phospholipase C” and “polypeptide having activity towards phosphatidylinositol (PI)” are used interchangeably. They relate to a polypeptide having activity towards phosphatidyl inositol (PI), meaning that its activity towards phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) is low compared to the PI activity. Pl-specific phospholipase C enzymes can either 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.

[0057] PC-, PE-, PA- and Pl-Specific Phospholipase C: The terms “PC-, PE-, PA,- and Pl-specific phospholipase C”, and “polypeptide having activity towards phosphatidylcholine (PC), phosphatidylethanoamine (PE), phosphatidic acid (PA) and phosphatidylinositol (PI)” are used interchangeably. They relate to a polypeptide having activity towards phosphatidylcholine (PC), phosphatidylethanoamine (PE), phosphatidic acid (PA), and phosphatidyl inositol (PI).

[0058] Phospholipase D: Phospholipase D (E.C. 3.1.4.4) is an enzyme acting on phospholipids such as lecithin and produces 1 ,2-diacylglycerophosphate and base group.

[0059] Protease: The term “protease” is defined herein as an enzyme that hydrolyses peptide bonds. It includes any enzyme belonging to the EC 3.4 enzyme group (including each of the thirteen subclasses thereof). The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including supplements 1- 5 published in Eur. J. Biochem. 223: 1-5 (1994); Eur. J. Biochem. 232: 1-6 (1995); Eur. J. Biochem. 237: 1-5 (1996); Eur. J. Biochem. 250: 1-6 (1997); and Eur. J. Biochem. 264: 610-650 (1999); respectively. The term "subtilases" refer to a sub-group of serine protease according to Siezen et al., 1991 , Protein Engng. 4: 719-737 and Siezen et al., 1997, Protein Science 6: 501-523. Serine proteases or serine peptidases is a subgroup of proteases characterised by having a serine in the active site, which forms a covalent adduct with the substrate. Further the subtilases (and the serine proteases) are characterised by having two active site amino acid residues apart from the serine, namely a histidine and an aspartic acid residue. The subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family. The term “protease activity” means a proteolytic activity (EC 3.4). Protease activity may be determined using methods described in the art (e.g., US 2015 / 0125925) or using commercially available assay kits (e.g., Sigma-Aldrich).

[0060] Soap stock: In the present contexts, “soap stock” refers to a fraction containing soaps, which is separated from the bulk of vegetable oil during a chemical refining process. The soaps are formed by reaction of a refining chemical, such as alkaline, with free fatty acids in the present in the vegetable oil. The exact composition of soap stocks depends on the vegetable oil source from which they are obtained; cottonseed soap stock, for instance, was found to be mainly composed of moisture and solvent, fatty acids, organic phosphates, monoglycerides, diglycerides, triglycerides, sterols, polyalcohols, carbohydrates and other miscellaneous components. The majority of these classes of organic compounds are found in soap stocks from other vegetable oils.

[0061] Soap stock and acid oil: Soap stock and acid oil are byproducts from processing vegetable oil. Soap stock is the result of neutralization of free fatty acids (FFA) in an oil with alkaline to saponify the FFA and separate it from the remaining oil phase. The soap stock is often neutralized with acid to recover the oil material including the FFA. This process produces acid oil.

[0062] Stoichiometric amount: The term “Stoichiometric amount” means, in effect, the measure of amount required for stoichiometry; i.e. the optimum amount where, assuming that the reaction proceeds to completion, all of the reagent is consumed, there is no deficiency of the reagent, and there is no excess of the reagent.

[0063] In the context of the invention “stoichiometric amount” refers in particular to the number of moles of a reagent (e.g. alkali, such as NaOH) added to a reaction mixture, which is equal to the number of moles of the compounds (e.g. free fatty acids and / or acid added as calcium chelating agent, such as citric acid) with which the reagent reacts in said reaction mixture. Transesterification: The term "transesterification" as used herein, refers to any of the following reactions: alcoholysis, acidolysis and interesterification.

[0064] Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1 ,4-beta-D-xylosidic linkages in xylans. Xylanase activity can be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mWI sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 pmole of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

[0065] Water degumming: The term “water degumming” refers to a process which involves treating crude oil with an amount of water to hydrate phospholipids present in the oil and make them separable by centrifugation.

[0066] DETAILED DESCRIPTION OF INVENTION

[0067] An aspect of the invention is a process for reducing phosphorus content containing components in an oil feedstock by phospholipase treatment, comprising contacting said oil feedstock under conditions sufficient for the enzymes to react with the phospholipids to form free fatty acid and lysophospholipid products. The lysophopholipid will dissolve in the aqueous phase resulting in reduction of the phosphorus content of the oil feedstock. The free fatty acids will stay in the oil.

[0068] The present invention relates to degumming of an oil feedstock, and to the finding that instead of applying a separate tank for acid degumming prior to enzymatic degumming an improved process can be achieved by combining acid and enzymatic treatment in the same tank / vessel.

[0069] The present invention, therefore, relates to a process for reducing phosphorous content in oil feedstock, said process comprising steps of: a. providing an oil feedstock; b. treating the oil feedstock with an acid; c. contacting the reaction mixture of step b) in presence of one or more phospholipases under condition allowing enzymatic hydrolysis of phospholipids in the oil feedstock; and d. separation of reaction mixture of step c), wherein the acid treatment and the enzymatic hydrolysis is performed in the same tank / vessel.

[0070] In one embodiment, the acidification and the enzymatic hydrolysis of the phospholipids is performed in one reaction vessel, and the acidification is performed before or during the enzymatic hydrolysis. Furthermore, the process has reduced residence time in the tank.

[0071] In one aspect of the present invention, oil feedstock comprises more than 50 ppm or more phosphorous content.

[0072] Oil typically will contain 50-250 ppm of phosphorus as phospholipid at the beginning of the treatment with the phospholipase.

[0073] In one aspect of the present invention, one or more phospholipases is selected from a group consisting of phospholipase A1 , phospholipase A2, phospholipase D, phospholipase B, lyso-phospholipase, phospholipase C, and combination thereof.

[0074] Phospholipases

[0075] The phospholipase may be any phospholipase that is suitable for the host cells and / or the methods described herein, such as a naturally occurring phospholipase (e.g., a native phospholipase from another species or an endogenous phospholipase expressed from a modified expression vector) or a variant thereof that retains phospholipase activity.

[0076] The phospholipase may be a bacterial phospholipase. For example, the phospholipase may be derived from a Gram-positive bacterium such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces, or a Gram-negative bacterium such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma.

[0077] In one embodiment, the phospholipase is derived from Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis.

[0078] In another embodiment, the phospholipase is derived from Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus.

[0079] In another embodiment, the phospholipase is derived from Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans.

[0080] The phospholipase may be a fungal phospholipase. For example, the phospholipase may be derived from a yeast such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia or Issatchenkia', or derived from a filamentous fungus such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria.

[0081] In another embodiment, the phospholipase is derived from Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis.

[0082] In another embodiment, the phospholipase is derived from Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0083] In an embodiment aspect, the phospholipase is an enzyme having phospholipase A activity. In an embodiment aspect, the phospholipase has 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% to SEQ ID NO: 1.

[0084] In an embodiment, the phospholipase A differs by no more than 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 as shown in SEQ ID NO: 1.

[0085] In a preferred embodiment the phospholipase A activity is at least 75% of the phospholipase A activity as shown in SEQ ID NO: 1.

[0086] The present invention preferably comprises or consists of the amino acid sequence of SEQ I D NO: 1 , or of an allelic variant thereof; or is a fragment thereof having phospholipase A activity.

[0087] In one embodiment of the present invention, said one or more phospholipases is provided in the form of a liquid, gel, paste, slurry or immobilized on a carrier or carrier particles.

[0088] In one embodiment of the present invention, said one or more phospholipases are dosed in a total amount corresponding to 0.02-30 mg enzyme protein / kg oil.

[0089] In one embodiment of the present invention, oil feedstock comprises crude oil and / or degummed oil. In a preferred embodiment the oil is selected from crude oil, water degummed oil, acid degummed oil and caustic refined oil.

[0090] In one embodiment of the present invention, the crude oil is plant oil, animal fat, algal oil, fish oil, waste oil, grease trap and any mixtures thereof.

[0091] In a preferred embodiment, the plant oil is selected from the group consisting of at least one of a rapeseed oil, a corn oil, a mustard oil, an olive oil, a palm oil, a palm kernel oil, a peanut oil, a safflower oil, a sesame oil, a soybean oil, a nut oil, a cottonseed oil, a crambe oil, a coconut oil, a meadowfoam oil, a vernonia oil, a iesquerella oil, a jatropha oil, a jojoba oil, a grape seed oil, a sunflower oil, and mixtures thereof.

[0092] In a preferred embodiment, the animal fat comprises cooking oil, yellow grease, tallow oil or choice white grease, poultry fat, fish oil or a blend thereof.

[0093] The phospholipase treatment can be carried out directly in the oil or after removal of slime (mucilage).

[0094] In an embodiment of the present invention, acid is added to the oil feedstock in the process stream prior to entry into the tank / vessel or added directly to the tank / vessel.

[0095] In an embodiment of the present invention, acid is added to the process stream prior to or at a mixing unit, such as a high shear mixer. In one embodiment, the acid is selected from a group consisting of phosphoric acid, acetic acid, citric acid, tartaric acid, oxalic acid, maleic acid, lactic acid and succinic acid, or a combination thereof.

[0096] A particular advantage of the present invention is that acid treatment of the oil feedstock as well as phospholipase treatment are performed in the same reaction tank / vessel, thereby reducing reaction time and leading to a simpler / cheaper plant setup. In a preferred embodiment the acid treatment is performed simultaneously with lipase treatment, however, performance as separate steps is also envisioned according to the invention.

[0097] In an embodiment of the present invention, step b) and step c) of the process are done for a duration of 2 hours or less, such as 1.5 hours or less, such as a duration of 5 minutes - 2 hours, such as 5 minutes to 1.5 hours or 5 minutes to 1 hour.

[0098] In an embodiment of the present invention, step b) and step c) are performed simultaneously or sequentially.

[0099] In an embodiment of the present invention, enzymatic hydrolysis of the phospholipids and the acidification are performed in the same vessel. That is, the enzymatic hydrolysis of the phospholipids is performed in a reaction vessel, and the acidification is performed during and / or before the enzymatic hydrolysis, in the same reaction vessel.

[0100] In an embodiment of the present invention, water is added to step c) of the process.

[0101] In an embodiment of the present invention, water content is in the range of 0.5 - 10% (w / w), such as in the range of 1-10% (w / w), in the range of 1-5% (w / w) or such as in the range of 0.5-5% (w / w).

[0102] In an embodiment of the present invention, pH range of process of the present invention is between 3.0 to about 9.0, preferably 3.5 to 8.0.

[0103] In a preferred embodiment, a suitable process comprises a) mixing acid with an oil to obtain an acidic mixture having pH of about 3.0 to 9.0, b) mixing a base with the acidic mixture to obtain a reacted mixture having pH of about 3.5-8.0, and c) degumming the reacted mixture with an enzyme of the present invention to obtain a degummed oil.

[0104] In an embodiment of the present invention, further comprises addition of alkali before step d). In an embodiment of the present invention, alkali is dosed in amounts which are stoichiometrically equivalent or in slight excess to the acid added in acidification / enzymatic reaction.

[0105] In an embodiments, the alkali (base) is selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium silicate, sodium carbonate, calcium carbonate, and a combination thereof.

[0106] In an embodiment of the present invention, alkali is incubated with reaction mixture of step c) from 1 minute to 5 hours, from 1 minute to 2 hours, from 5 minutes to 2 hours.

[0107] In an embodiment of the present invention, one or more chelating agents capable of complexing Ca and / or Mg ions is added prior to contacting the oil feedstock with the one or more phospolipase.

[0108] In an embodiment of the present invention, chelating agent is selected from a group consisting of EDTA, EGTA, tri-sodium phosphate, sodium tripolyphosphate, or a combination thereof.

[0109] The process of the invention may be performed as a batch process, or as a continuous process. Thus, the process can fit into existing process setup whether it is a batch operation or the typical continuous process used in the industry.

[0110] In an embodiment of the present invention, separation the mixture of step c) into light and heavy phase.

[0111] In an embodiment of the present invention, separation is performed using gravity separation, a decanter, a centrifuge, a separator, membranes, and any combinations thereof.

[0112] In an embodiment of the present invention, separated light phase is further processed by bleaching to reduce the phosphorous further before it is used for production of hydrotreated vegetable oil (HVO), hydroprocessed esters and fatty acids (HEFA), renewable diesel (RD), aviation biokerosene and / or sustainable aviation fuel (SAF).

[0113] In an embodiment of the present invention, separated light phase is further processed by distillation to reduce the phosphorous further before it is used for production of hydrotreated vegetable oil (HVO), hydroprocessed esters and fatty acids (HEFA), renewable diesel (RD), aviation biokerosene and / or sustainable aviation fuel (SAF).

[0114] In an embodiment of the present invention, separated heavy phase is subjected to chemical refining to separate gums and / or soapstock. In an embodiment of the present invention, process for producing renewable diesel and / or diesel and / or oleochemicals comprising use of a process as described above.

[0115] The invention is further described in the following numbered paragraphs.

[0116] Paragraph 1. A process for reducing phosphorous content in oil feedstock, said process comprising steps of: a. providing an oil feedstock; b. treating the oil feedstock with an acid; c. contacting the reaction mixture of step b) in presence of one or more phospholipases under condition allowing enzymatic hydrolysis of phospholipids in the oil feedstock; and d. separation of reaction mixture of step c), wherein the acid treatment and the enzymatic hydrolysis is performed in the same tank / vessel.

[0117] Paragraph 2. The process according to paragraph 1 , wherein the oil feedstock comprises more than 50 ppm or more phosphorous content.

[0118] Paragraph 3. The process according to paragraph 1 , wherein one or more enzymes phospholipases is selected from a group consisting of phospholipase A1 , phospholipase A2, phospholipase D, phospholipase B, lyso-phospholipase, phospholipase C, and combination thereof.

[0119] Paragraph 4. The process according to paragraph 1 , wherein the one or more phospholipases is provided in the form of a liquid, gel, paste, slurry or immobilized on a carrier or carrier particles.

[0120] Paragraph 5. The process according to any of the preceding paragraphs, wherein said one or more phospholipases are dosed in a total amount corresponding to 0.02-30 mg enzyme protein / kg oil.

[0121] Paragraph 6. The process according to paragraph 1 , wherein the oil feedstock comprises crude oil and / or degummed oil. Paragraph 7. The process according to paragraph 6, wherein the crude oil is plant oil, animal fat, algal oil, fish oil, waste oil, grease trap and any mixtures thereof.

[0122] Paragraph 8. The process according to paragraph 6, wherein the plant oil is selected from the group consisting of at least one of a rapeseed oil, a corn oil, a mustard oil, an olive oil, a palm oil, a palm kernel oil, a peanut oil, a safflower oil, a sesame oil, a soybean oil, a nut oil, a cottonseed oil, a crambe oil, a coconut oil, a meadowfoam oil, a vernonia oil, a iesquerella oil, a jatropha oil, a jojoba oil, a grape seed oil, a sunflower oil, and mixtures thereof.

[0123] Paragraph 9. The process according to paragraph 6, wherein the animal fat comprises cooking oil, yellow grease, tallow oil or choice white grease, poultry fat, fish oil or a blend thereof.

[0124] Paragraph 10. The process of paragraph 1 , wherein the acid is added to the oil feedstock in the process stream prior to entry into the tank / vessel or added directly to the tank / vessel.

[0125] Paragraph 11. The process of paragraph 10, wherein the acid is added to the process stream prior to or at a mixing unit, such as a high shear mixer.

[0126] Paragraph 12. The process according to paragraph 1, wherein said step b) and step c) are done for a duration of 2 hours or less, such as 1 .5 hours or less, such as a duration of 5 minutes - 2 hours, such as 5 minutes to 1.5 hours or 5 minutes to 1 hour.

[0127] Paragraph 13. The process according to paragraph 1 , wherein the acid is selected from a group consisting of phosphoric acid, acetic acid, citric acid, tartaric acid, oxalic acid, maleic acid, lactic acid and succinic acid, or a combination thereof.

[0128] Paragraph 14. The process according to paragraph 1 , wherein one or more chelating agents capable of complexing Ca and / or Mg ions is added prior to contacting the oil feedstock with the one or more phospholipases. Paragraph 15. The process according to paragraph 14, wherein the chelating agent is selected from a group consisting of EDTA, EGTA, tri-sodium phosphate, sodium tripolyphosphate, or a combination thereof.

[0129] Paragraph 16. The process according to any of the preceding paragraphs, wherein water is added to step c).

[0130] Paragraph 17. The process according to paragraph 16, wherein the water content is in the range of 0.5 - 10% (w / w), such as in the range of 1-10% (w / w), in the range of 1-5% (w / w) or such as in the range of 0.5-5% (w / w).

[0131] Paragraph 18. The process according to paragraph 1 , wherein step c) further comprises contacting the oil with an enzyme selected from a group consisting of cellulases, endoglucanases, phospholipases, lipases, cutinases, esterases, cellobiohydrolases, xylanase, chlorophyllase, mannanases, hemicellulases, pectinases, proteases, phytases, and combination thereof.

[0132] Paragraph 19. The process according to any of the preceding paragraphs, wherein at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 60 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent at least about 97 percent, at least about 98 percent, at least about 99 percent of the original content of phosphorous in the oil feedstock is reduced.

[0133] Paragraph 20. The process according to paragraph 1 , wherein said process is conducted at a pH in the range of 3.0 to 9.0, preferably in the range of 3.5 to 8.0.

[0134] Paragraph 21. The process according to paragraph 1 , wherein the said process is conducted at a temperature, which is in the range of 45-90°C.

[0135] Paragraph 22. The process according to paragraph 1 , further comprises addition of alkali before step d). Paragraph 23. The process according to paragraph 22, wherein the alkali is dosed in amounts which are stoichiometrically equivalent or in slight excess to the acid added in acidification / enzymatic reaction.

[0136] Paragraph 24. The process according to paragraphs 22-23, wherein the alkali is selected from sodium hydroxide, potassium hydroxide, sodium carbonate and combinations thereof.

[0137] Paragraph 25. The process according to paragraphs 22-24, wherein said alkali is incubated with reaction mixture of step c) from 1 minute to 5 hours, from 1 minute to 2 hours, from 5 minutes to 2 hours.

[0138] Paragraph 26. The process according to any of the preceding paragraphs, wherein separating the mixture of step c) into light and heavy phase.

[0139] Paragraph 27. The process according to any of the preceding paragraphs, wherein separation is performed using gravity separation, a decanter, a centrifuge, a separator, membranes, and any combinations thereof.

[0140] Paragraph 28. The process according to any of the preceding paragraphs, wherein the separated light phase is further processed by bleaching to reduce the phosphorous further before it is used for production of hydrotreated vegetable oil, hydroprocessed esters and fatty acids (HEFA), renewable diesel (RD), aviation biokerosene and / or sustainable aviation fuel (SAF).

[0141] Paragraph 29. The process according to any of the preceding paragraphs, wherein the separated light phase is further processed by distillation to reduce the phosphorous further before it is used for production of hydrotreated vegetable oil, hydroprocessed esters and fatty acids (HEFA), renewable diesel (RD), aviation biokerosene and / or sustainable aviation fuel (SAF).

[0142] Paragraph 30. The process according to paragraph 27, wherein the separated heavy phase is subjected to chemical refining to separate gums and / or soapstock. Paragraph 31. The process according to paragraph 1, wherein the step b) and step c) are performed simultaneously or sequentially.

[0143] Paragraph 33. The process according to paragraph 1 , further comprises addition of bleaching clay.

[0144] Paragraph 32. A process for producing renewable diesel and / or diesel and / or oleochemicals comprising use of a process of any of the preceding paragraphs.

[0145] The present invention is further described by the following example that should not be construed as limiting the scope of the invention.

[0146] EXAMPLES

[0147] Example 1. Degumming of soybean oil and waste oils blends

[0148] Degumming was assessed by total phosphorus which was measured by Inductively coupled plasma optical emission spectrometry (ICP-OES). Oil blends were prepared in the ratios indicated in Table 1. Citric acid (50% solution) was added to the oil at the ratios indicated. After addition of acid to oil (30g), the tubes were agitated at 70°C briefly by hand for 2 minutes to allow for pH equilibration prior to addition of phospholipase of SEQ ID NO: 1. Phospholipase of SEQ ID NO: 1 at a dose of 0.25 ppm and 3% water based on the amount of oil was added to samples. Samples were shaken and then incubated at 60°C while being agitated using a rotator at 40 rpm. Reaction times ranged from 15 minutes to 240 minutes. The phospholipase of SEQ ID NO: 1 was then deactivated by incubation at 90°C in a water bath, and then centrifuged at 1800xg for 3 minutes.

[0149] The phosphorous, calcium, magnesium and iron composition in the oils used in the experiment is indicated in Table 2. For blend 1 and 2, a treatment of phospholipase of SEQ ID NO: 1 for 15 minutes incubation time performs better than the acid chelation treatments for 30 and 60 minutes (see Table 2). In addition, a 15-minute incubation with phospholipase of SEQ ID NO: 1 was required for phosphorus removal compared to an incubation at 240 minutes for blend 1 and 2.

[0150] Table 1. Composition of oil blends.

[0151] Table 2. Metal composition of oils before and after degumming treatments measured by ICP-OES.

[0152] Example 2: Enzymatic degumming of soybean oil and blends with animal fats and waste oils assisted by sodium hydroxide for pH adjustment

[0153] Degumming was assessed by total phosphorus which was measured by Inductively coupled plasma optical emission spectrometry (ICP-OES). Oil blends were prepared in the ratios indicated in Table 1. Citric acid (50% solution) was added to the oil in dry basis at the ratios indicated in Table 3. After addition of acid to oil (30g), the tubes were agitated at 60°C briefly by hand for 2 minutes to allow for pH equilibration prior to addition of phospholipase of SEQ ID NO: 1. Phospholipase of SEQ I D NO: 1 at a dose of 0.25 ppm and 3% water based on the amount of oil was added to samples. Samples were shaken and then incubated at 60°C while being agitated using a rotator at 40 rpm. Reaction times ranged from 15 minutes to 60 minutes. After the allotted time for degumming, a 20% sodium hydroxide solution was added in dry basis to each tube (15.6 pl to treatments with 200 ppm citric acid, 50 pl to treatments with 600 ppm citric acid, and 75 pl to treatments with 1000 ppm citric acid) and the tubes were incubated for three minutes at 60°C. The phospholipase of SEQ ID NO: 1 was then deactivated by incubation at 90°C in a water bath, and then centrifuged at 1800xg for 3 minutes.

[0154] The phosphorous, calcium, magnesium and iron composition in the oils used in the experiment is indicated in Table 3. For blend 1 , 2 and 100% soy oil, a treatment of phospholipase of SEQ ID NO: 1 for 15 minutes incubation time performs better than the acid chelation treatments at 650 and 1000 ppm (see Table 3) particularly for phosphorus removal. Table 3. Metal composition of oils before and after degumming treatments measured by ICP-OES. Example 3: Comparison of acid and enzymatic degumming with and without sodium hydroxide addition.

[0155] Degumming was assessed by total phosphorus which was measured by Inductively coupled plasma optical emission spectrometry (ICP-OES). A representative oil blend nominally composed of 60% soybean oil, 20% tallow, 10% distillers corn oil, 5% yellow grease and 5% used cooking oil was degummed. Citric acid (50% solution) was added to the oil in dry basis at the ratios indicated in Table 4. After addition of acid to oil (30g), the tubes were agitated at 60°C briefly by hand for 2 minutes to allow for pH equilibration prior to addition of phospholipase of SEQ ID NO: 1. Phospholipase of SEQ ID NO: 1 at a dose of 0.25 ppm and 3% water based on the amount of oil was added to samples. Samples were shaken and then incubated at 60°C while being agitated using a rotator at 40 rpm for 40 minutes. After the allotted time for degumming, a 10% sodium hydroxide solution was added in dry basis to each tube (10.9 pl to treatments with 225 ppm citric acid, 57 pl to treatments with 1100 ppm citric acid) as indicated in Table 3 and the tubes were incubated for 15 minutes at 60°C. The phospholipase of SEQ ID NO: 1 was then deactivated by incubation at 90°C in a water bath, and then centrifuged at 1800xg for 3 minutes. All treatments were exposed to the same process steps (time and temperature) whether or not they included caustic or enzyme.

[0156] The phosphorous, calcium, magnesium and iron composition in the oils used in the experiment is indicated in Table 4. The treatments including phospholipase of SEQ ID NO: 1 provided the best phosphorus reduction, with the combination of phospholipase of SEQ ID NO: 1 being the best of all four treatments.

[0157] Table 4. Metal composition of oils before and after degumming treatments measured by ICP-OES.

[0158] Example 4: Treatment of degummed oils with bleaching clay Degumming and bleaching were assessed by total phosphorus which was measured by Inductively coupled plasma optical emission spectrometry (ICP-OES). Oil blends were prepared in the ratios indicated in Table 5. Citric acid (50% solution) was added to the oil in dry basis at the ratios indicated in Table 6. After addition of acid to oil (250g), the vessels were agitated at 60°C briefly using an overhead mixer for 2 minutes to allow for pH equilibration prior to addition of phospholipase of SEQ ID NO: 1. Phospholipase of SEQ ID NO: 1 at a dose of 0.25 ppm and 3% water based on the amount of oil was added to samples. Samples were then incubated at 60°C while being agitated using an overhead mixer at 120 rpm for 30 minutes. The phospholipase of SEQ ID NO: 1 was then deactivated by incubation at 85°C in a water bath, and then centrifuged at 1800xg for 3 minutes. All treatments were exposed to the same process steps (time and temperature) whether or not they included caustic or enzyme.

[0159] For bleaching, 30g of degummed oil was preheated to 85°C in a 50 ml flask while agitating with a stir bar at 275 rpm. Bleaching clay was added in the amounts indicated in Table 6. Flasks were then heated at 100-105 °C on a stir plate, stirring at 270 rpm for 30 minutes and at an absolute pressure of 354 mbar. After bleaching, samples were filtered through two sheets of filter paper (Whatman 42 neutral / non-charged 47 mm diameter CAT No. 1442-047) to remove the clay from the oil.

[0160] The phosphorous, calcium, magnesium and iron composition in the oils used in the experiment is indicated in T able 6. For the treatments including phospholipase of SEQ ID NO: 1 only 1.2% (w / w) bleaching clay to oil was required to reduce the phosphorus content of the oil below 4 ppm for all three oil blends tested. For the oils treated with acid degumming greater than 1.2% (w / w) bleaching was needed to achieve the same specification of less than 4 ppm phosphorus.

[0161] Table 5. Composition of oil blends. Table 6. Metal composition of oils before and after degumming treatments measured by ICP-OES.

Claims

Claims:

1. A process for reducing phosphorous content in oil feedstock, said process comprising steps of: a. providing an oil feedstock; b. treating the oil feedstock with an acid; c. contacting the reaction mixture of step b) in presence of one or more phospholipases under condition allowing enzymatic hydrolysis of phospholipids in the oil feedstock; and d. separation of reaction mixture of step c), wherein the acid treatment and the enzymatic hydrolysis is performed in the same tank / vessel.

2. The process according to claim 1, wherein the oil feedstock comprises more than 50 ppm of phosphorous content.

3. The process according to claim 1 , wherein one or more enzymes phospholipases is selected from a group consisting of phospholipase A1 , phospholipase A2, phospholipase D, phospholipase B, lyso-phospholipase, phospholipase C, and combination thereof.

4. The process according to claim 1, wherein the one or more enzymes phospholipases is provided in the form of a liquid, gel, paste, slurry or immobilized on a carrier or carrier particles.

5. The process according to any of the preceding claims, wherein said one or more phospholipases are dosed in a total amount corresponding to 0.02-30 mg enzyme protein / kg oil.

6. The process according to claim 1 , wherein the oil feedstock comprises crude oil and / or degummed oil.

7. The process according to claim 6, wherein the crude oil is plant oil, animal fat, algal oil, fish oil, waste oil, grease trap and any mixtures thereof.

8. The process according to claim 6, wherein the plant oil is selected from the group consisting of at least one of a rapeseed oil, a corn oil, a mustard oil, an olive oil, a palm oil, a palm kernel oil, a peanut oil, a safflower oil, a sesame oil, a soybean oil, a nut oil, a cottonseed oil, a crambe oil, a coconut oil, a meadowfoam oil, a vernonia oil, a iesquerella oil, a jatropha oil, a jojoba oil, a grape seed oil, a sunflower oil, and mixtures thereof.

9. The process according to claim 6, wherein the animal fat comprises cooking oil, yellow grease, tallow oil or choice white grease, poultry fat, fish oil or a blend thereof.

10. The process of claim 1 , wherein the acid is added to the oil feedstock in the process stream prior to entry into the tank / vessel or added directly to the tank / vessel.

11. The process of claim 10, wherein the acid is added to the process stream prior to or at a mixing unit, such as a high shear mixer.

12. The process according to claim 1 , wherein said step b) and step c) are done for a duration of 2 hours or less, such as 1.5 hours or less, such as a duration of 5 minutes - 2 hours, such as 5 minutes to 1 .5 hours or 5 minutes to 1 hour.

13. The process according to claim 1 , wherein the acid is selected from a group consisting of phosphoric acid, acetic acid, citric acid, tartaric acid, oxalic acid, maleic acid, lactic acid and succinic acid, or a combination thereof.

14. The process according to claim 1 , wherein one or more chelating agents capable of complexing Ca and / or Mg ions is added prior to contacting the oil feedstock with the one or more phospholipases.

15. The process according to claim 14, wherein the chelating agent is selected from a group consisting of EDTA, EGTA, tri-sodium phosphate, sodium tripolyphosphate, or a combination thereof.

16. The process according to any of the preceding claims, wherein water is added to step c).

17. The process according to claim 16, wherein the water content is in the range of 0.5 - 10% (w / w), such as in the range of 1-10% (w / w), in the range of 1-5% (w / w) or such as in the range of 0.5-5% (w / w).

18. The process according to claim 1 , wherein step c) further comprises contacting the oil with an enzyme selected from a group consisting of cellulases, endoglucanases, cellobiohydrolases, xylanase, phospholipases, lipases, cutinases, esterases, chlorophyllase, mannanases, hemicellulases, pectinases, proteases, phytases, and combination thereof.

19. The process according to any of the preceding claims, wherein at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 60 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent at least about 97 percent, at least about 98 percent, at least about 99 percent of the original content of phosphorous in the oil feedstock is reduced.

20. The process according to claim 1 , wherein said process is conducted at a pH in the range of 3.0 to 9.0, preferably in the range of 3.5 to 8.0.

21. The process according to claim 1 , wherein the said process is conducted at a temperature, which is in the range of 45-90°C.

22. The process according to claim 1 , further comprises addition of alkali before step d).

23. The process according to claim 22, wherein the alkali is dosed in amounts which are stoichiometrically equivalent or in slight excess to the acid added in acidification / enzymatic reaction.

24. The process according to claims 22-23, wherein the alkali is selected from sodium hydroxide, potassium hydroxide, sodium carbonate and combinations thereof.

25. The process according to claims 22-24, wherein said alkali is incubated with reaction mixture of step c) from 1 minute to 5 hours, from 1 minute to 2 hours, from 5 minutes to 2 hours.

26. The process according to any of the preceding claims, wherein separating the mixture of step c) into light and heavy phase.

27. The process according to any of the preceding claims, wherein separation is performed using gravity separation, a decanter, a centrifuge, a separator, membranes, and any combinations thereof.

28. The process according to any of the preceding claims, wherein the separated light phase is further processed by bleaching to reduce the phosphorous further before it is used for production of hydrotreated vegetable oil, hydroprocessed esters and fatty acids (HEFA), renewable diesel (RD), aviation biokerosene and / or sustainable aviation fuel (SAF).

29. The process according to any of the preceding claims, wherein the separated light phase is further processed by distillation to reduce the phosphorous further before it is used for production of hydrotreated vegetable oil, hydroprocessed esters and fatty acids (HEFA), renewable diesel (RD), aviation biokerosene and / or sustainable aviation fuel (SAF).

30. The process according to claim 26, wherein the separated heavy phase is subjected to chemical refining to separate gums and / or soapstock.

31. The process according to claim 1 , wherein the step b) and step c) are performed simultaneously or sequentially.

32. A process for producing renewable diesel and / or diesel and / or oleochemicals comprising use of a process of any of the preceding claims.

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