Process for refining a plant oil

Abstract

In one aspect, provided herein is a process for refining a plant oil, comprising a step of contacting the oil with an enzyme which is capable of hydrolysing chlorophyll or a chlorophyll derivative, wherein the enzyme is contacted with the oil in the presence of at least 0.1% by weight phospholipid.

Description

1 / 92 “PROCESS FOR REFINING A PLANT OIL” Field of Invention

[001] The present invention relates to the industrial processing of plant-derived food and feed products, especially vegetable oils. The invention can be employed to reduce or eliminate contamination by chlorophyll and chlorophyll derivatives. Background of the Invention

[002] Chlorophyll is a green pigment widely found throughout the plant kingdom. Chlorophyll is essential for photosynthesis and is one of the most abundant organic metallic compounds found on Earth. Thus, many plant-derived products, including food and animal feed, contain significant amounts of chlorophyll.

[003] For example, vegetable oils derived from oilseeds such as soybeans, palm or rapeseed (canola), cottonseed and peanut oil typically contain some chlorophyll. However, the presence of high levels of chlorophyll pigments in vegetable oils is generally undesirable. This is because chlorophyll imparts an undesirable green color and can induce oxidation of the oil during storage, leading to oil deterioration.

[004] Several methods have been employed to remove chlorophyll from vegetable oils. Chlorophyll can be removed during many stages of the oil production process, including seed crushing, oil extraction, degumming, caustic treatment, and bleaching steps. However, the bleaching step is often the most significant for reducing chlorophyll residues to an acceptable level. During bleaching, the oil is heated and passed through an adsorbent to remove chlorophyll and other color-bearing compounds that influence the appearance and / or stability of the finished oil. The adsorbent used in Petition 870260073365, dated 07 / 23 / 2026, page 8 / 198 Step 2 / 92 of bleaching is typically clay.

[005] In the edible oil processing industry, the use of such steps typically reduces chlorophyll levels in the processed oil to between 0.02 and 0.05 ppm. However, the bleaching step increases processing costs and reduces oil yield due to carryover in the bleaching clay. The use of clay can remove many desirable compounds such as carotenoids and tocopherol from the oil. Also, the use of clay is expensive, and this is particularly due to the treatment with used clay (i.e., the waste) which can be difficult, dangerous (tending to auto-ignition) and therefore costly to handle. Thus, attempts have been made to remove chlorophyll from the oil by other means, for example, through the use of the enzyme chlorophyllase.

[006] In plants, chlorophyllase (chlase) is believed to be involved in the degradation of chlorophyll and catalyzes the hydrolysis of an ester bond in chlorophyll to yield chlorophyllide and phytol. The document in WO 2006009676 describes an industrial process in which chlorophyll contamination can be reduced in a composition such as a plant oil by treatment with chlorophyllase. The water-soluble chlorophyllide that is produced in the process is also green in color, but can be removed by aqueous extraction or treatment with silica.

[007] Chlorophyll is frequently partially degraded in seeds used for oil production as well as during oil extraction from the seeds. A common modification is the loss of the magnesium ion from the porphyrin ring (chlorin) to form the derivative known as pheophytin (see Figure 1). The loss of the highly polar magnesium ion from the porphyrin ring results in significantly different physicochemical properties of pheophytin compared to chlorophyll. Typically, pheophytin is more abundant in the oil during processing than chlorophyll. Pheophytin has Petition 870260073365, dated 07 / 23 / 2026, page 9 / 198 3 / 92 a greenish color and can be removed from oil by a process analogous to that used for chlorophyll, for example, as described in WO 2006009676 by an esterase reaction catalyzed by an enzyme that has pheophytinase activity. Under certain conditions, some chlorophyllases are capable of hydrolyzing pheophytin as well as chlorophyll, and thus are suitable for removing both contaminants. The products of pheophytin hydrolysis are the red / brown pheophorbide and phytol. Pheophorbide can also be produced by the loss of a magnesium ion from chlorophyllide, i.e., following chlorophyll hydrolysis (see Figure 1). WO 2006009676 teaches the removal of pheophorbide by a method analogous to that of chlorophyllide, for example, by aqueous extraction or silica adsorption.

[008] Pheophytin can be further degraded to pyropheophytin, either by the activity of plant enzymes during harvesting or by storage of oilseeds or by processing conditions (e.g., heat) during oil refining (see Behavior of Chlorophyll Derivatives in Canola Oil Processing, JAOCS, Volume no. 9 (September 1993), pages 837–841). One possible mechanism is the enzymatic hydrolysis of the methyl ester linkage of the isocyclic ring of pheophytin followed by non-enzymatic conversion of the unstable intermediate to pyropheophytin. The 28–29 kDa enzyme from Chenopodium album called pheophorbidase is reportedly capable of catalyzing an analogous reaction in pheophorbidin, to produce the phytol-free derivative of pyropheophytin known as pyropheophorbidin (see Figure 26).Pyropheophorbides are less polar than pheophorbides, resulting in pheophorbides having decreased water solubility and decreased oil solubility compared to pheophorbides.

[009] Depending on the processing conditions, the Petition 870260073365, dated 07 / 23 / 2026, page 10 / 198 4 / 92 Pyropheophytin may be more abundant than both pleiophytin and chlorophyll in vegetable oils during processing (see Table 9 in volume 2.2 of Bailey's "Industrial Oil and Fat Products" (2005), 6th edition, edited by Fereidoon Shahidi. John Wiley & Sons). This is partly due to the loss of magnesium from chlorophyll during harvesting and storage of plant material. If extended heat treatment at 90°C or above is used, the amount of pyropheophytin in the oil will likely increase and may be higher than the amount of pheophytin. Chlorophyll levels are also reduced by heating oilseeds before pressing and extraction, as well as by oil degumming and alkali treatment during the refining process. It has also been observed that phospholipids in the oil can complex with magnesium, thus reducing the amount of chlorophyll.Thus, chlorophyll is a relatively minor contaminant compared to pyropheophytin (and pheophytin) in many plant oils.

[010] There is still a need for an improved process to remove chlorophyll and chlorophyll derivatives such as pheophytin and pyropheophytin from plant oils. In particular, there is a need for a process in which chlorophyll and chlorophyll derivatives are removed with marked efficiency, while reducing the loss of other desirable compounds from the oil. Brief Description of the Invention

[011] In one aspect, the present invention provides a process for refining a plant oil, comprising a step of contacting the oil with an enzyme capable of hydrolyzing chlorophyll or a chlorophyll derivative, wherein the enzyme is contacted with the oil in the presence of at least 0.1% by weight of phospholipid.

[012] In some embodiments, the enzyme is brought into contact with the oil in the presence of less than 0.2% by weight of lysophospholipid, for Petition 870260073365, dated 07 / 23 / 2026, page 11 / 198 5 / 92 example, less than 0.15%, less than 0.1% or less than 0.05% by weight, based on the total weight of oil.

[013] In one embodiment, the enzyme is brought into contact with the oil before degumming the oil. In another embodiment, the enzyme is contacted with the oil during an oil degumming step. The degumming step may comprise, for example, degumming with water, degumming with acid, enzymatic degumming, and / or total degumming / neutralization (e.g., addition of an acid to an oil followed by neutralization with an alkali).

[014] When the enzyme is brought into contact with the oil before degumming, in particular embodiments, an enzymatic degumming step may comprise bringing the oil into contact with a phospholipase (e.g., phospholipase A1, phospholipase A2, or phospholipase C) or an acyltransferase. The acyltransferase may comprise, for example, the amino acid sequence SEQ ID NO: 23 or a sequence that has at least 80% sequence identity with it.

[015] In another embodiment, the process comprises contacting the oil with the enzyme and a phospholipase that does not produce lysopholides (e.g., phospholipase C) in a single step. For example, the enzyme can be contacted with the oil during an enzymatic degumming step using phospholipase C, i.e., the enzyme and phospholipase C are used simultaneously.

[016] In further embodiments, the enzyme is brought into contact with the oil in the presence of at least 0.5%, at least 1%, at least 1.5% or at least 2% by weight of phospholipid.

[017] In further embodiments, the enzyme is brought into contact with the oil at a temperature of less than 80°C, preferably less than 70°C, preferably from 55°C to 65°C, preferably from Petition 870260073365, dated 07 / 23 / 2026, p. 12 / 198 6 / 92 58° to 62°C, for example, about 60°C. Preferably, the enzyme is brought into contact with the oil in the presence of 1 to 5% by weight of water, for example, about 1% or about 2% by weight of water. In one embodiment, the enzyme is brought into contact with the oil at a pH of 6.0 to 6.8, for example, 6.3 to 6.5.

[018] Preferably, the process does not include a clay treatment step. The process preferably additionally includes performing a deodorizing step to produce a deodorized oil and a distillate (e.g., an aqueous distillate or a nitrogenous distillate). Typically, the process produces a level of carotenoids and / or tocopherol in the refined oil and / or distillate (deodorized or non-deodorized) that is high compared to a process that includes a clay treatment step.

[019] In one embodiment, the enzyme comprises a chlorophyllase, pheophytinase, pyropheophytinase, or pheophytin pheophorbide hydrolase. For example, the enzyme may comprise a polypeptide sequence as defined in any of the SEQ ID NOs: 1, 2, 4, 6, or 8 to 15, or a functional fragment or variant thereof. Preferably, the enzyme comprises a polypeptide sequence that has at least 75% sequence identity to any of the SEQ ID NOs: 1, 2, 4, 6, or 8 to 15, for example, over at least 50 amino acid residues. In particular, in preferred embodiments, the enzyme comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or a sequence that has at least 90% sequence identity with them.

[020] In another aspect, the invention provides a refined plant oil obtainable by a process as defined above.

[021] In a further aspect, the invention provides a distillate (for example, an aqueous or nitrogenous distillate) obtainable by the process Petition 870260073365, dated 07 / 23 / 2026, page 13 / 198 7 / 92 as defined above, that is, a process as described herein comprising a deodorizing step.

[022] In a further aspect, the invention provides a process as defined above, for increasing the level of carotenoids and / or tocopherol in a refined oil and / or a distillate obtained by deodorizing the oil.

[023] As described in this document, the activity of chlorophyllases and related enzymes in oil has been found to be dependent on the phospholipid content of the oil. Therefore, in one embodiment, the present invention provides an enhanced oil refining process in which chlorophyllase or a related enzyme is used in the presence of a minimum level of phospholipid. Furthermore, based on the demonstration that high levels of lysophospholipid are associated with reduced chlorophyllase activity, in one embodiment, the invention provides an enhanced process in which the enzyme is used in the presence of a low level of lysophospholipid. By enhancing the enzyme activity, the process of the present invention advantageously facilitates the removal of chlorophyll and chlorophyll derivatives, typically without the need for a clay treatment step.This can increase the level of beneficial compounds such as tocopherol and carotenoids in the oil, which can be recovered in a deodorizing step or retained in the finished product. Brief Description of the Drawings

[024] Figure 1 shows the reactions involving chlorophyll and derivatives and enzymes used in the present invention.

[025] Figure 2 shows the amino acid sequence of Arabidopsis thaliana chlorophyllase (SEQ ID NO: 1).

[026] Figure 3 shows the amino acid sequence of Triticum aestivum chlorophyllase (SEQ ID NO: 2). Petition 870260073365, dated 07 / 23 / 2026, page 14 / 198 8 / 92

[027] Figure 4 shows a nucleotide sequence that encodes Triticum aestivum chlorophyllase (SEQ ID NO: 3).

[028] Figure 5 shows the amino acid sequence of Chlamydomonas reinhardtii chlorophylase (SEQ ID NO: 4).

[029] Figure 6 shows the nucleotide sequence that encodes Chlamydomonas reinhardtii chlorophyllase (SEQ ID NO: 5).

[030] Figure 7 shows the amino acid sequence of a pheophytin pheophorbide hydrolase (PPH) from Arabidopsis thaliana (SEQ ID NO: 6). A chloroplast transit peptide is shown in bold.

[031] Figure 8 shows the nucleotide sequence of an Arabidopsis thaliana cDNA encoding pheophytin pheophorbidin hydrolase (SEQ ID NO: 7). PPH of SEQ ID NO: 6 is encoded by residues 173 to 1627 of SEQ ID NO: 7.

[032] Figure 9 shows the polypeptide sequence of Populus trichocarpa PPH (SEQ ID NO: 8).

[033] Figure 10 shows the polypeptide sequence of Vitis vinifera PPH (SEQ ID NO: 9).

[034] Figure 11 shows the polypeptide sequence of Ricinus communis PPH (SEQ ID NO: 10).

[035] Figure 12 shows the polypeptide sequence of Oryza sativa (japonica cultivar group) PPH (SEQ ID NO: 11).

[036] Figure 13 shows the polypeptide sequence of Zea mays PPH (SEQ ID NO: 12).

[037] Figure 14 shows the polypeptide sequence of Nicotiana tabacum PPH (SEQ ID NO: 13).

[038] Figure 15 shows the Oryza sativa japonica PPH group polypeptide sequence (SEQ ID NO: 14).

[039] Figure 16 shows (a) the polypeptide sequence of Petition 870260073365, dated 07 / 23 / 2026, page 15 / 198 9 / 92 Physcomitrella patens subsp. patens PPH (SEQ ID NO: 15)

[040] Figure 17 schematically shows the fusion of the wheat (Triticum aestivum) chlorophyllase gene into the aprE signaling sequence.

[041] Figure 18 schematically shows the pBN-TRI CIIL plasmid containing the wheat (Triticum aestivum) chlorophyllase gene.

[042] Figure 19 schematically shows the fusion of the Chlamydomonas reinhardtii chlorophyllase gene into the aprE signaling sequence.

[043] Figure 20 schematically shows the pBNCHL_CHL plasmid containing the Chlamydomonas reinhardtii chlorophyllase gene.

[044] Figure 21 shows samples of refined rapeseed oil treated with chlorophyllase in the presence of different surfactants, which include soy lecithin, sorbitan monooleate and sorbitan trioleate, as described in Example 3.

[045] Figure 22 shows samples of refined oil or a mixture of refined oil and crude soybean oil, with or without chlorophyllase treatment and chlorophyll addition, as described in Example 4.

[046] Figure 23 shows the relative fluorescence values ​​from HPLC analysis indicative of pheophytin levels in rapeseed oil samples following treatment with chlorophyllase in the presence of varying levels of lecithin and modified lecithin (modified by an acyltransferase), as described in Example 5.

[047] Figure 24 shows the relative fluorescence values ​​from HPLC analysis indicative of pheophorbide levels in rapeseed oil samples following treatment with chlorophyllase in the presence of varying levels of lecithin and modified lecithin (modified by an acyltransferase), as described in Example 5.

[048] Figure 25 shows the relative fluorescence values ​​from HPLC analysis indicative of pyropheophytin levels in the oil samples. Petition 870260073365, dated 07 / 23 / 2026, page 16 / 198 10 / 92 rapeseed following treatment with chlorophyllase in the presence of varying levels of lecithin and modified lecithin (modified by an acyltransferase), as described in Example 5.

[049] Figure 26 shows the relative fluorescence values ​​from HPLC analysis indicative of pyropheophorbide levels in rapeseed oil samples following treatment with chlorophyllase in the presence of varying levels of lecithin and modified lecithin (modified by an acyltransferase), as described in Example 5.

[050] Figure 27 shows the relative fluorescence values ​​from HPLC analysis indicative of pheophytin levels in rapeseed oil samples following treatment with Triticum aestivum or Chlamydomonas reinhardtii chlorophyllase in the presence of an acyltransferase, phospholipase C or phospholipase A1, as described in Example 6.

[051] Figure 28 shows an HPLC chromatogram using absorbance detection (430 nm) indicating numbered peaks associated with: 1 = chlorophyllide b; 2 = chlorophyllide a; 3 = neoxanthin; 3' = neoxanthin isomer; 4 = neochrome; 5 = violaxanthin; 6 = luteoxanthin; 7 = auroxanthin; 8 = antheraxanthin; 8' = antheraxanthin isomer; 9 = mutatoxanthin; 10 = lutein; 10' = lutein isomer; 10' = lutein isomer; 11 = pheophorbide b; 12 = pheophorbide a; 13 = chlorophyll b; 13' = chlorophyll b'; 14 = chlorophyll a; 14' = chlorophyll a'; 15 = pheophytin b; 15' = pheophytin b'; 16 =e-carotene; 17 = pheophytin a; 17' - pheophytin a'; 18 = pyropheophytin b; 19 = pyropheophytin a.

[052] Figure 29 shows pheophytin ae and pyropheophytin levels in the oil at various stages of a standard refining process using clay bleaching and an enzymatic refining process using chlorophyllase without clay treatment, as described in Example 8.

[053] Figure 30 shows the levels of pheophorbid and pyrophypheophorbid in the oil at various stages of a standard refining process. Petition 870260073365, dated 07 / 23 / 2026, page 17 / 198 11 / 92 through the use of clay bleaching and an enzymatic refining process using chlorophyllase without clay treatment, as described in Example 8.

[054] Figure 31 is a diagrammatic representation of an oil refining process according to an embodiment of the present invention.

[055] Figure 32 shows the amino acid sequence of a mature lipid acyltransferase (GCAT) from mutant Aeromonas salmonicida with an Asn80Asp mutation after undergoing post-translational modification (SEQ ID No. 23).

[056] Figure 33 shows the effect of chlorophyllase on the degradation of pheophytin a in the oil.

[057] Figure 34 shows the effect of pH and % water on chlorophyllase activity.

[058] Figure 35 shows epimeric forms of pheophytin and their reorganization.

[059] Figure 36 shows the relative ratio of pheophytin a epimers at different pH after a reaction time of 4 hours.

[060] Figure 37 shows the effect of temperature on pheophytin hydrolysis by treating oil with chlorophyllase.

[061] Figure 38 shows the effect of temperature on pyropheophytin levels.

[062] Figure 39 shows the effect of temperature on total chlorophyll and pheophytin and pyropheophytin levels.

[063] Figure 40 shows the effect of different mixing conditions on pheophytin hydrolysis.

[064] Figure 41 shows pheophytin as a function of time and pH in oil treated with chlorophyllase.

[065] Figure 42 shows the ratio of pheophytin isomer to as Petition 870260073365, dated 07 / 23 / 2026, page 18 / 198 12 / 92 is a function of time and pH in oil treated with chlorophyllase.

[066] Figure 43 shows pyropheophytin as a function of time and pH in oil treated with chlorophyllase.

[067] Figure 44 shows enzymatic pyropheophytin hydrolysis as a function of time and pH in chlorophyllase-treated oil.

[068] Figure 45 shows the effect of pH on chlorophyllase degradation of pheophytin.

[069] Figure 46 shows the effect of pH on the amount of pheophytin ae epimers a'.

[070] Figure 47 shows the effect of pH on iropheophytin levels.

[071] Figure 48 shows the effect of pH on total pheophytin and pyropheophytin levels.

[072] Figure 49 shows the effect of pH on pheophorbidin levels.

[073] Figure 50 shows the effect of pH on pheophytin in rapeseed oil after 2 hours of chlorophyllase treatment in the water degumming (WDG) and total degumming (TDG) processes.

[074] Figure 51 shows the effect of water content and pH on pheophytin in oil treated with chlorophyllase.

[075] Figure 52 shows the effect of water content and pH on pyropheophytin in oil treated with chlorophyllase.

[076] Figure 53 shows the effect of water content and pH on pheophytin epimers in oil treated with chlorophyllase.

[077] Figure 54 shows the effect of temperature, pH adjustment and reaction time on pheophytin levels by different dosages of chlorophyllase oil treatment.

[078] Figure 55 shows the effect of temperature, pH adjustment and reaction time on pyropheophytin levels at different dosages of Petition 870260073365, dated 07 / 23 / 2026, page 19 / 198 13 / 92 oil treatment with chlorophyllase.

[079] Figure 56 shows the effect of temperature, pH adjustment and reaction time on pheophytin a epimer levels at different dosages of chlorophyllase oil treatment. Detailed Description of the Invention

[080] In one aspect the present invention relates to a process for refining a plant oil. Typically, the process is used to remove chlorophyll and / or chlorophyll derivatives from the oil, or to reduce the level of chlorophyll and / or chlorophyll derivatives in the oil, for example, where chlorophyll and / or chlorophyll derivatives are present as a contaminant. Chlorophyll and Chlorophyll Derivatives

[081] Chlorophyll derivatives typically mean compounds comprising both a porphyrin (chlorin) ring and a phytol group (end end), including magnesium-free phytol-containing derivatives such as pheophytin and pyropheophytin. Chlorophyll and chlorophyll derivatives (containing phytol) typically have a greenish color as a result of the porphyrin (chlorin) ring present in the molecule. The loss of magnesium from the porphyrin ring means that pheophytin and pyropheophytin have a more brownish color than chlorophyll. Thus, the presence of chlorophyll and chlorophyll derivatives in an oil can impart an undesirable green, greenish, or brownish color to that oil. In one embodiment, the present process can be carried out for the purpose of removing or reducing the green or brown coloration present in the oil. Consequently, the present process can be referred to as a bleaching or decolorization process.

[082] The enzymes used in the process can hydrolyze chlorophyll and chlorophyll derivatives containing phytol to cleave the phytol end of the chlorin ring. The hydrolysis of chlorophyll and chlorophyll derivatives typically results in compounds such as chlorophyllide, pheophorbide and Petition 870260073365, dated 07 / 23 / 2026, page 20 / 198 14 / 92 pyropheophorbides are phytol-free derivatives of chlorophyll. These compounds still contain the color-bearing porphyrin ring, as chlorophyllide is green and pheophorbides and pyropheophorbides have a reddish-brown color. In some embodiments, it may also be desirable to remove this phytol-free derivative and to reduce the green / red / brown coloration in the oil. Thus, in one embodiment of the invention, the process may further comprise a step of removing or reducing the level of phytol-free chlorophyll derivative in the oil. The process may involve bleaching or decolorization to remove the green and / or red / brown coloration from the oil.

[083] Chlorophyll or chlorophyll derivatives can be either forms a or b. Thus, as used in this document, the term “Chlorophyll” includes chlorophyll a and chlorophyll b. Similarly, both forms a and b are covered when this document refers to pheophytin, pyropheophytin, chlorophyllide, pheophorbidin and pyropheophorbidin. Plant Oils

[084] Any plant oil can be treated according to the present process for the purpose of removing undesirable contamination by chlorophyll and / or chlorophyll derivatives. The oil can be derived from any type of plant, and from any part of a plant, including whole plants, leaves, stems, flowers, roots, plant protoplasts, seeds, and plant cells and their progeny. The class of plants from which the products can be treated in the method of the invention includes higher plants, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. This includes plants of a variety of ploidy levels, including polyploid, diploid, haploid, and hemizygous states.

[085] In preferred embodiments, the oil may comprise a vegetable oil, including processed oils from oilseeds or oilseeds (for example, seed oils such as canola (seed) Petition 870260073365, dated 07 / 23 / 2026, page 21 / 198 15 / 92 rapeseed) oil and fruit oils such as palm oil). Examples of suitable oils include rice bran, soybean, canola (rapeseed), palm, olive, cottonseed, corn, palm kernel, coconut, peanut, sesame or sunflower oil. The process of the invention can be used in conjunction with methods for processing essential oils, for example, fruit seed oils, for example, grape seed, apricot, borage, etc. The process of the invention can be used in conjunction with methods for processing highly phosphorous oils (for example, a soybean oil). Preferably the oil is a crude plant oil. Chlorophyll and Chlorophyll Derivatives in Oil

[086] Chlorophyll and / or chlorophyll derivatives (e.g., chlorophyll, pheophytin and / or pyropheophytin) may be present in the oil naturally, as a contaminant, or as an undesirable component in a processed product. Chlorophyll and / or chlorophyll derivatives (e.g., chlorophyll, pheophytin and / or pyropheophytin) may be present at any level in the oil. Typically, chlorophyll, pheophytin and / or pyropheophytin may be present as a natural contaminant in the oil at a concentration of 0.001 to 1,000 mg / kg (0.001 to 1,000 ppm, 10⁻⁷ to 10⁻¹% by weight), based on the total weight of the oil. In further embodiments, chlorophyll and / or chlorophyll derivatives may be present in the oil at a concentration of 0.1 to 100, 0.5 to 50, 1 to 50, 1 to 30, or 1 to 10 mg / kg, based on the total weight of the oil.

[087] The chlorophyll-free derivative of phytols may also be present in the oil. For example, chlorophyllide, pyrophorebidin and / or pyrophorebidin may be present at any level in the oil. Typically, chlorophyllide, pyrophorebidin and / or pyrophorebidin may be present in the oil, either before or after treatment with an enzyme according to the method of the present invention, at a concentration of 0.001 to 1,000 mg / kg (from 0.001 Petition 870260073365, dated 07 / 23 / 2026, page 22 / 198 16 / 92 to 1,000 ppm. 10-7 to 10-1% by weight), based on the total weight of the oil. In additional embodiments, chlorophyllide, pyrophorebidin and / or pyrophorebidin may be present in the composition at a concentration of 0.1 to 100, 0.5 to 50, 1 to 50, 1 to 30 or 1 to 10 mg / kg, based on the total weight of the composition. Enzymes that hydrolyze chlorophyll or a chlorophyll derivative.

[088] The process of the present invention comprises the step of contacting the oil with an enzyme capable of hydrolyzing chlorophyll or a chlorophyll derivative. Typically, hydrolyzing chlorophyll or a chlorophyll derivative means hydrolyzing an ester linkage in chlorophyll or a chlorophyll derivative (containing phytol), for example, to cleave a phytol group from the chlorin ring in chlorophyll or the chlorophyll derivative. Thus, the enzyme typically has esterase or hydrolase activity. Preferably, the enzyme has esterase or hydrolase activity in an oil phase, and optionally also in an aqueous phase.

[089] Thus, the enzyme may, for example, be a chlorophyllase, pheophytinase, or pyropheophytinase. Preferably, the enzyme is capable of hydrolyzing at least one, at least two, or all three of chlorophyll, pheophytin, and pyropheophytin. In a particularly preferred embodiment, the enzyme has chlorophyllase, pheophytinase, and pyropheophytinase activity. In further embodiments, two or more enzymes may be used in the method, each enzyme having a different substrate specificity. For example, the method may comprise the combined use of two or three enzymes selected from a chlorophyllase, a pheophytinase, and a pyropheophytinase.

[090] Any polypeptide that has an activity that can hydrolyze chlorophyll or a chlorophyll derivative can be used as the enzyme in the process of the invention. By enzyme is meant to encompass any polypeptide that has hydrolytic activity on chlorophyll or a derivative. Petition 870260073365, dated 07 / 23 / 2026, page 23 / 198 17 / 92 of chlorophyll, including, for example, enzyme fragments, etc. Any isolated, recombinant, synthetic, or chimeric polypeptide (or a combination of synthetic and recombinant) may be used. Enzyme Activity Assay (Chlorophyllase, Pheophytinase or PlROPHEOPHYTINASE).

[091] Hydrolytic activity on chlorophyll or a chlorophyll derivative can be detected using any suitable assay technique, for example, based on an assay described in this document. For example, hydrolytic activity can be detected using fluorescence-based techniques. In a suitable assay, a polypeptide to be tested for hydrolytic activity on chlorophyll or a chlorophyll derivative is incubated in the presence of a substrate, and product or substrate levels are monitored by fluorescence measurement. Suitable substrates include, for example, chlorophyll, pheophytin and / or pyropheophytin. Products that can be detected include chlorophyllide, pheophorbide, pyropheophorbide and / or phytol.

[092] Test methods for detecting hydrolysis of chlorophyll or a chlorophyll derivative are presented in, for example, AH Khamessan et al. (1994), Journal of Chemical Technology & Biotechnology, 60(1), pages 73 to 81; Klein and Vishniac (1961), I. Biol. Chem. 236: 2,544 to 2,547; and Kiani et al. (2006), Analytical Biochemistry 353: 93 to 98.

[093] Alternatively, a suitable assay may be based on HPLC detection and quantification of substrate or product levels following the addition of a putative enzyme, for example, based on the techniques described below. In one embodiment, the assay may be performed as described in Horncro-Mendez et al. (2005), Food Research International 38 (8-9): 1067-1072. In another embodiment, the following assay may be used: Petition 870260073365, dated 07 / 23 / 2026, page 24 / 198 18 / 92

[094] 170 μl mM of HEPES, pH 7.0, is added to 20 μl of 0.3 mM chlorophyll, pheophytin, or pyropheophytin dissolved in acetone. The enzyme is dissolved in 50 mM HEPES, pH 7.0. 10 μl of enzyme solution is added to 190 μl of substrate solution to initiate the reaction and incubated at 40°C for various periods of time. The reaction was stopped by the addition of 350 μl of acetone. Then, centrifugation (2 minutes at 18,000 g) of the supernatant was analyzed by HPLC, and the amounts of (i) chlorophyll and chlorophyllide (ii) pheophytin and pheophorbide or (iii) pyropheophytin and pyropheophorbide were determined.

[095] One enzyme activity unit is defined as the amount of enzyme that hydrolyzes one micromole of substrate (e.g., chlorophyll, pheoophytin or pyropheophytin) per minute at 40°C, for example, in an assay method as described in this document.

[096] In preferred embodiments, the enzyme used in the present method was chlorophyllase, pheophytinase and / or pyropheophytinase activity of at least 1,000 U / g, at least 5,000 U / g, at least 10,000 U / g, or at least 50,000 U / g, based on activity units per gram of purified enzyme, for example, as determined by an assay method described in this document. Chlorophylases

[097] In one embodiment, the enzyme is capable of hydrolyzing at least chlorophyll. Any polypeptide that catalyzes the hydrolysis of a chlorophyll ester bond to yield chlorophyllide and phytol can be used in the process. For example, a chlorophyllase, chlase, or chlorophyll chlorophyllo-hydrolase, or a polypeptide that has similar activity (e.g., chlorophyll-chlorophyllo-hydrolase 1 or chlase 1, or chlorophyll chlorophyllo-hydrolase 2 or chlase 2; see, for example, NCBI P59677-1 and P59678, respectively) can be Petition 870260073365, dated 07 / 23 / 2026, page 25 / 198 19 / 92 used in the process.

[098] In one embodiment the enzyme is a chlorophyllase classified by the enzyme nomenclature classification (e.g., 3.1.1.14). Any isolated, recombinant or synthetic or chimeric (a combination of synthetic and recombinant) polypeptide (e.g., enzyme or catalytic antibody) may be used, see, for example, Marchler-Bauer (2003) Nucleic Acids Res. 31: 383 to 387. In one aspect, chlorophyllase may be an enzyme as described in the document in WO 0229022 or in the document in WO 2006009676. For example, Arabidopsis thaliana chlorophyllase may be used as described, for example, in NCBI entry M 123753. Thus, chlorophyllase may be a polypeptide comprising the sequence SEQ ID NO: 1 (see Figure 2). In another embodiment, chlorophyllase is derived from algae, for example, Phaeodactylum tricornutum.

[099] In another embodiment, chlorophyllase is derived from wheat, for example, from Triticum sp., especially from Triticum aestivum. For example, chlorophyllase may be a polypeptide comprising the sequence SEQ ID NO: 2 (see Figure 3), or it may be encoded by the nucleotide sequence SEQ ID NO: 3 (see Figure 4).

[0100] In another embodiment, chlorophyllase is derived from Chlamydomonas sp., especially Chlamydomonas reinhardtii. For example, chlorophyllase may be a polypeptide comprising the sequence SEQ ID NO: 4 (see Figure 5), or it may be encoded by the nucleotide sequence SEQ ID NO: 5 (see Figure 6). Pheophytin Pheophorbide Hydrolase

[0101] In one embodiment, the enzyme is capable of hydrolyzing pheophytin and pyropheophytin. For example, the enzyme may be pheophytinase or pheophytin pheophorbide hydrolase (PPH), for example, an enzyme as described in Schelbert et al., The Plant Cell 21: 767 to 785 (2009). Petition 870260073365, dated 07 / 23 / 2026, p. 26 / 198 20 / 92

[0102] PPH and related enzymes are capable of hydrolyzing pyropheophytin in addition to pheophytin. However, PPH is inactive in chlorophyll. As described in Schelbert et al., PPH orthologs are commonly present in eukaryotic photosynthetic organisms. PPHs represent a defined subgroup of α / β hydrolases that are phylogenetically distinct from chlorophyllases; the two groups are distinct in terms of sequence homology and substrates.

[0103] In specific embodiments of the invention, the enzyme can be any known PPH derived from any species or a functional variant or fragment thereof, or it can be derived from any known PPH enzyme. For example, in one embodiment, the enzyme is a PPH from Arabidopsis thaliana, for example, a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 (see Figure 7), or a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 7 (see Figure 8, NCBI accession number NP_196884, GenBank ID no. 15240707), or a functional variant or fragment thereof.

[0104] In further embodiments, the enzyme may be a PPH derived from any of the following species: Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera, Oryza sativa, Zea mays, Nicotiana tabacum, Ostreococcus lucimarinus, Ostreococcus taurii, Physcomitrella patens, Phaeodactylum tricornutum, Chlamydomonas reinhardtii, or Micromonas sp. RCC299. For example, the enzyme may be a polypeptide comprising an amino acid sequence, or is encoded by a nucleotide sequence, defined in one of the following database entries shown in Table 1, or a functional fragment or variant thereof: Table 1 Organism Accession Genbank ID Arabidopsis thaliana NP_196884 15240707 Populus trichocarpa XP_002314066 224106163 Petition 870260073365, dated 07 / 23 / 2026, p. 27 / 198 21 / 92 Organism Access Genbank ID Vitis vinifera CAO40741 157350650 Oryza sativa (japonica) NP_001057593 115467988 Zea mays ACF87407 194706646 Nicotiana tabacum CA099125 156763846 Ostreococcus lucimarinus XP_00141 5589 145340970 Ostrcococcus tauri CAL50341 116000661 Organism Access Genbank ID Physcomitrella patens XP_001761725 168018382 Phacodaetylum tricomutum XP 002181821 219122997 Chlamydomonas reinardtii XP_001702982 159490010 Micromonas sp RCC299 ACO62405 226516410

[0105] For example, the enzyme may be a polypeptide as defined in any of the SEQ ID NOs 8 to 15 (Figures 9 to 16), or a functional fragment or variant thereof. Variants and Fragments

[0106] Variants and functional fragments of known sequences that hydrolyze chlorophyll or a chlorophyll derivative can also be employed in the present invention. "Functional" means that the fragment or variant retains detectable hydrolytic activity on chlorophyll or a chlorophyll derivative. Typically, such variants and fragments show homology to a known chlorophyllase, pheophytinase, or pyropheophytinase sequence, for example, at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of sequence identity to a known chlorophyllase, pheophytinase, or pyropheophytinase amino acid sequence, for example, to SEQ ID NO: 1 or any of SEQ ID NOs: 1, 2, 4, 6, or 8 to 15, for example, in a region of at least about 10, 20, 30, 50, 100, 200, 300, 500, or 1,000 or more residues, or the entire length of the sequence.

[0107] The percentage of sequence identity can be determined by analysis with a sequence comparison algorithm or by visual inspection. In one aspect, the comparison algorithm Petition 870260073365, dated 07 / 23 / 2026, page 28 / 198 The 22 / 92 sequence is a BLAST algorithm, for example, version 2.2.2 of the BLAST algorithm.

[0108] Other enzymes having chlorophyllase, pheophytinase and / or pyropheophytinase activity suitable for use in the process can be identified by determining the presence of conserved sequence motifs present, for example, in known chlorophyllase, pheophytinase or pyropheophytinase sequences. For example, conserved sequence motifs revealed in PPH enzymes include the following: LPGFGVG (SEQ ID NO: 16), DFLGQG (SEQ ID NO: 17), GNSLGG (SEQ ID NO: 18), LVKGVTLLNATPFW (SEQ ID NO: 19), HPAA (SEQ ID NO: 20), EDPW (SEQ ID NO: 21), and SPAGHCPH (SEQ ID NO: 22). In some embodiments, an enzyme for use in the present invention may comprise one or more of these sequences. The GNSLGG motif (SEQ ID NO: 18) contains an active site serine residue. Polypeptide sequences that have suitable activity can be identified by searching genome databases, for example, the microbiome metagenome database (JGI-DOE, USA), for the presence of these motifs. Enzyme Isolation and Production

[0109] Enzymes for use in the present invention may be isolated from their natural sources or may be, for example, produced using recombinant DNA techniques. Nucleotide sequences encoding polypeptides that have chlorophyllase, pheophytinase and / or pyropheophytinase activity may be isolated or constructed and used to produce the corresponding polypeptides.

[0110] For example, a genomic DNA and / or cDNA library can be constructed using chromosomal DNA or messenger RNA from the organism that produces the polypeptide. If the sequence of Petition 870260073365, dated 07 / 23 / 2026, page 29 / 198 If the 23 / 92 amino acid of the polypeptide is known, identified oligonucleotide probes can be synthesized and used to identify polypeptide-coding clones from the genomic library prepared from the organism. Alternatively, the identified oligonucleotide probe containing sequences homologous to another known polypeptide gene can be used to identify polypeptide-coding clones. In the latter case, lower rigor hybridization and washdown conditions are used.

[0111] Alternatively, polypeptide-encoding clones can be identified by inserting genomic DNA fragments into an expression vector, such as a plasmid, transforming enzyme-negative bacteria with the resulting genomic DNA library, and then coating the transformed bacteria in agar containing an enzyme inhibited by the polypeptide, thus allowing clones expressing the polypeptide to be identified.

[0112] Also in a further alternative, the nucleotide sequence encoding the polypeptide can be prepared synthetically by established standard methods, for example, the phosphoramidite method described by Beucage SL et al (1981) Tetrahedron Letters 22, pages 1859 to 1869, or by the method described by Matthes et al (1984) EMBO J. 3, pages 801 to 805. In the phosphoramidite method, oligonucleotides are synthesized, for example, in an automated DNA synthesizer, purified, annealed, ligated and cloned into appropriate vectors.

[0113] The nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with Petition 870260073365, dated 07 / 23 / 2026, page 30 / 198 24 / 92 standard techniques. Each linked fragment corresponds to several parts of the entire nucleotide sequence. The DNA sequence can also be prepared by polymerase chain reaction (PGR) using specific primers, for example, as described in US 4,683,202 or in Saiki RK et al (Science (1988) 239, pages 487 to 491).

[0114] The term “nucleotide sequence” as used in this document refers to an oligonucleotide sequence or polynucleotide sequence, and variants, homologs, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic, synthetic or recombinant origin, and may be double-stranded or single-stranded if it represents the sense and antisense strands.

[0115] Typically, the nucleotide sequence encoding a polypeptide that has chlorophyllase, pheophytinase and / or pyropheophytinase activity is prepared using recombinant DNA techniques. However, in an alternative embodiment of the invention, the nucleotide sequence can be synthesized, in whole or in part, using chemical methods well known in the art (see, Caruthers MH et al. (1980) Nuc Acids Res Symp Ser 215 to 23 and Horn T et al (1980) Nuc Acids Res Symp Ser 225 to 232). Enzyme Sequence Modification

[0116] Once an enzyme-coding nucleotide sequence has been isolated, or a putative enzyme-coding nucleotide sequence has been identified, it may be desirable to modify the selected nucleotide sequence, for example, it may be desirable to mutate the sequence to prepare an enzyme according to the present invention.

[0117] Mutations can be introduced using synthetic oligonucleotides. These oligonucleotides contain sequences of Petition 870260073365, dated 07 / 23 / 2026, page 31 / 198 25 / 92 nucleotides flanking the desired mutation sites. A suitable method is revealed in Morinaga et al. (Biotechnology (1984) 2, pages 646 to 649). Another method of introducing mutations into enzyme-coding nucleotide sequences is described in Nelson and Long (Analytical Biochemistry (1989), 180, pages 147 to 151).

[0118] Instead of site-directed mutagenesis, as described above, an individual can introduce mutations randomly, for example, using a commercial kit such as the GeneMorph PGR mutagenesis kit from Stratagene, or the random PCR diversification mutagenesis kit from Clontech. EP 0 583 265 refers to PGR-based mutagenesis enhancement methods, which can also be combined with the use of mutagenic DNA analogs such as those described in EP 0 866 796. Error-prone PGR technologies are suitable for producing variants of chlorophyll-hydrolyzing enzymes and / or chlorophyll derivatives with preferred characteristics. WO0206457 refers to the molecular evolution of lipases.

[0119] A third method for obtaining new sequences is to fragment non-identical nucleotide sequences, either using any number of restriction enzymes or a single enzyme such as DNase I, and reassemble complete nucleotide sequences that encode functional proteins. Alternatively, an individual may use one or multiple non-identical nucleotide sequences and introduce mutations during the reassembly of the complete nucleotide sequence. DNA scrambling and family scrambling technologies are suitable for producing enzyme variants with preferred characteristics. Suitable methods for performing 'scrambling' can be disclosed in EP 0752008, EP 1138763, EP1103606. Scrambling can also be combined with other forms of DNA mutagenesis as described in US Petition 870260073365, dated 07 / 23 / 2026, page 32 / 198 26 / 92 6,180,406 and WO 01 / 34835.

[0120] In this way, it is possible to produce numerous random and site-directed mutations in a nucleotide sequence, either in vivo or in vitro, and subsequently screen for enhanced functionality of the encoded polypeptide by various means. Using silicon- and exo-mediated recombination methods (see, WO 00 / 58517, US 6,344,328, US 6,361,974), for example, molecular evolution can be performed where the variant produced retains very low homology to known enzymes or proteins. Such variants then obtained may have significant structural analogy to known chlorophyllase, pheophytinase, or pyropheophytinase enzymes, but have very low amino acid sequence homology.

[0121] As a non-limiting example, furthermore, natural and mutational variations of a polynucleotide sequence can be recombined either with the wild type or other natural and mutational variations to produce new variants. Such new variants can also be screened for enhanced functionality of the encoded polypeptide.

[0122] The application of the molecular evolution methods mentioned above allows the identification and selection of variants of the enzymes of the present invention that have preferred characteristics without any prior knowledge of protein structure and function, and allows the production of unpredictable but beneficial mutations or variants. There are numerous examples of the application of molecular evolution in the art for the optimization or alteration of enzymatic activity; such examples include, but are not limited to, one or more of the following: optimized expression and / or activity in a host cell or increased enzymatic activity in vitro, altered substrate and / or product specificity, increased or decreased enzymatic stability or structural changes, altered enzymatic activity / specificity under environmental conditions. Petition 870260073365, dated 07 / 23 / 2026, p. 33 / 198 27 / 92 preferred factors, for example, temperature, pH, substrate.

[0123] As will be apparent to those skilled in the art, using molecular evolution tools, an enzyme can be altered to enhance the enzyme's functionality. Appropriately, a nucleotide sequence encoding an enzyme (e.g., a chlorophyllase, pheophytinase, and / or pyropheophytinase) used in the invention may encode a variant enzyme, that is, the variant enzyme may contain at least one amino acid substitution, deletion, or addition compared to a parental enzyme. Variant enzymes retain at least 1%, 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 99% identity with the parental enzyme. Suitable parental enzymes may include any enzyme with hydrolytic activity on chlorophyll and / or a chlorophyll derivative. Polypeptide Sequences

[0124] The present invention also encompasses the use of amino acid sequences encoded by a nucleotide sequence encoding a pyropheophytinase for use in any of the methods and / or uses of the present invention.

[0125] As used in this document, the term amino acid sequence is synonymous with the term polypeptide and / or the term protein. In some cases, the term amino acid sequence is synonymous with the term peptide. The amino acid sequence can be prepared / isolated from a suitable source, or it can be synthetically produced, or it can be prepared using recombinant DNA techniques. Suitablely, amino acid sequences can be obtained from the isolated polypeptides taught in this document by standardized techniques.

[0126] A suitable method for determining amino acid sequences of isolated polypeptides is as follows. The purified polypeptide can be lyophilized and 100 pg of the lyophilized material can be Petition 870260073365, dated 07 / 23 / 2026, page 34 / 198 28 / 92 dissolved in 50 μL of a mixture of 8 M urea and 0.4 M ammonium bicarbonate, pH 8.4. The dissolved protein can be denatured and reduced for 15 minutes at 50°C after overcooking with nitrogen and adding 5 μL of 45 mM dithiothreitol. After cooling to room temperature, 5 μL of 100 mM iodoacetamide can be added to the cysteine ​​residues, which will be derivatized for 15 minutes at room temperature in the dark under nitrogen.

[0127] 135 μL of water and 5 μg of Lys-C endoproteinase in 5 μL of water can be added to the above reaction mixture and digestion can be carried out at 37°C under nitrogen for 24 hours. The remaining peptides can be separated by reverse-phase HPLC on a C18 VYDAC tank (0.46 x 15 cm; 10; The Separation Group, California, USA) using sokente A: 0.1% TFA in water and sokente B: 0.1% TFA in acetonitrHa. Selected peptides can be rechromatographed on a DevetosH C18 chromatography panel using the same soker system, prior to N-terminal sequencing. Sequencing can be performed using a 476A sequencer from AppHed Biosystems with fast liquid sequencers prepared according to the manufacturer's instructions (AppHed Biosystems, California, USA). Sequence Comparison

[0128] Currently, the term homologous means a whole that has a certain homotology with the amino acid sequences in question and the nucleotide sequences in question. Currently, the term homology can be equated with identity. The homotogamous amino acid sequence and / or nucleotide sequence must provide and / or encode a polypeptide that retains functional activity and / or enhances enzymatic activity.

[0129] In the present context, a homotomous sequence is taken Petition 870260073365, dated 07 / 23 / 2026, p. 35 / 198 29 / 92 to include an amino acid sequence that may be at least 75, 85, or 90% identical, preferably at least 95 or 98% identical to the sequence in question. Typically, homologs will comprise the same active sites etc. as the amino acid sequence in question. Although homology may also be considered in terms of similarity (i.e., amino acid residues that have similar chemical properties / functions), in the context of the present invention, it is preferred to express homology in terms of sequence identity.

[0130] In the present context, a homologous sequence is taken to include the nucleotide sequence that may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to a nucleotide sequence encoding a polypeptide of the present invention (the sequence in question). Typically, homologs will comprise the same sequences encoding the active sites etc. as the amino acid sequence in question. Although homology may also be considered in terms of similarity (i.e., amino acid residues that have similar chemical properties / functions), in the context of the present invention, it is preferred to express homology in terms of sequence identity.

[0131] Homology comparisons can be conducted by looking, or more commonly, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of homology between two or more sequences. The percentage of homology can be calculated on continuous sequences, that is, one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called a gapless alignment. Typically, such Petition 870260073365, dated 07 / 23 / 2026, page 36 / 198 30 / 92 gap-free alignments are performed only on a relatively small number of residues.

[0132] Although it is a very simple and consistent method, it fails to consider that, for example, in a pair of otherwise identical sequences, an insertion or deletion will cause the following amino acid residues to be placed out of alignment, thus potentially resulting in a wide reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce excellent alignments that take into account possible insertions and deletions without unduly penalizing the global homology score. This is achieved by inserting gaps in the sequence alignment to try to maximize local homology.

[0133] However, these more complex methods assign gap penalties to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible—reflecting higher relatedness between the two compared sequences—achieves a higher score than one with many gaps. Affine gap costs are typically used to carry a relatively high cost for the existence of a gap and a lower penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will clearly produce optimized alignments with fewer gaps. Most alignment programs allow gap penalties to be modified. However, it is preferred to use the default values ​​when using such software for sequence comparisons.

[0134] Calculating the maximum percentage of homology therefore requires, first and foremost, the production of an excellent alignment, which leads to Petition 870260073365, dated 07 / 23 / 2026, p. 37 / 198 31 / 92 consideration of gap penalties. A suitable computer program for performing such alignment is Vector NTI Advance™ 11 (Invitrogen Corp.). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see, Ausubel et al 1999 Short Protocols in Molecular Biology, Fourth Edition - Chapter 18), and FASTA (Altschul et al 1990 J. Mol. 403 to 410). Both BLAST and FASTA are available for online and offline research (see, Ausubel et al 1999, pages 7 to 58 and 7 to 60). However, for some applications, it is preferred to use the Vector NTI Advance™ 11 program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol Lett 1999 174(2): 247-50; and FEMS Microbiol Lett 1999 177(1): 187-8).

[0135] Although the final homology percentage can be measured in terms of identity, the alignment process alone is not typically based on an all-or-nothing pair comparison. Instead, a scaled similarity scoring matrix is ​​generally used to assign scores to each pair comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is ​​the BLOSUM62 matrix – the standard matrix for the BLAST suite of programs. Vector NTI programs generally use either the public default values ​​or a custom symbol comparison table, if provided (see the user manual for further details). For some applications, it is preferred to use the default values ​​for the Vector NTI Advance™11 package.

[0136] Alternatively, percentage of homologies can be calculated using the multiple alignment features in Vector NTI Advance™11 (Invitrogen Corp.), based on an algorithm analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244). Once the software has produced an excellent alignment, it is possible to calculate % of Petition 870260073365, dated 07 / 23 / 2026, page 38 / 198 32 / 92 homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0137] Gap penalties should be used during sequence identity determination, and then preferably, the default parameters for the program are used for pairwise alignment. For example, the following parameters are the current default parameters for pairwise alignment for BLAST 2: Table 2 FOR BLAST2 DNA Protein Expected threshold 10 10 Word size 11 3 Scoring parameters Equivalent / non-equivalent scores 2, -3 n / a Matrix n / a BLOSUM62 Gap costs Existence: 5 Extension: 2 Existence: 11 Extension: 1

[0138] In one embodiment, preferably the sequence identity for the nucleotide sequences and / or amino acid sequences can be determined using BLAST2 (blastn) with the scoring parameters adjusted as defined above.

[0139] For the purposes of the present invention, the degree of identity is based on the number of sequence elements that are the same. The degree of identity according to the present invention for amino acid sequences can be adequately determined by means of computer programs known in the art such as Vector NTI Advance™11 (Invitrogen Corp.). For pairwise alignment, the scoring parameters used are preferably BLOSUM62 with a gap existence penalty of 11 and a gap extension penalty of 1.

[0140] Appropriately, the degree of identity in relation to Petition 870260073365, dated 07 / 23 / 2026, page 39 / 198 33 / 92 A nucleotide sequence is defined as consisting of at least 20 consecutive nucleotides, preferably at least 30 consecutive nucleotides, preferably at least 40 consecutive nucleotides, preferably at least 50 consecutive nucleotides, preferably at least 60 consecutive nucleotides, or preferably at least 100 consecutive nucleotides. If appropriate, the degree of identity with respect to a nucleotide sequence can be determined over the entire sequence. Amino Acid Mutations

[0141] Sequences may also have deletions, insertions, or substitutions of amino acid residues that produce a silent alteration and result in a substance of equivalent functionality. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues while the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups that have similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0142] Conservative substitutions can be made, for example, according to the table below. Amino acids in the same block in the second column, and preferably in the same row in the third column, can be substituted for each other: Table 3 Aliphatic Nonpolar GAP ILV Petition 870260073365, dated 07 / 23 / 2026, p. 40 / 198 34 / 92 Polar - Uncharged CSTM NQ Polar - Charged DE KR Aromatic HFWY

[0143] The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the exchange of an amino acid residue with an alternative residue) which may occur, i.e., like-for-like substitution such as basic-for-basic, acidic-for-acid, polar-for-polar, etc. Non-homologous substitution may also occur, i.e., from one class of residue to another or alternatively involving the inclusion of non-natural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyrylalanine, thienylalanine, naphthylalanine and phenylglycine. Replacements may also be made by non-natural amino acids.

[0144] Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence, including alkyl groups such as methyl, ethyl, or propyl groups in addition to amino acid spacers such as glycine or β-alanine residues. A further form of variation involving the presence of one or more amino acid residues in peptoid form will be well understood by those skilled in the art. For avoidance of doubt, peptoid form is used to refer to variant amino acid residues in which the α-carbon substituent group is on the nitrogen atom of the residue rather than the α-carbon. Processes for preparing peptides in peptoid form are known in the art, for example, Simon RJ et al., PNAS (1992) 89(20), 9367 to 9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132 to 134. Petition 870260073365, dated 07 / 23 / 2026, p. 41 / 198 35 / 92 Nucleotide Sequences

[0145] Nucleotide sequences for use in the present invention or encoding a polypeptide having the specific properties defined herein may include synthetic or modified nucleotides. A number of different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate chains and / or the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the present invention, it should be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be performed to enhance the in vivo activity or lifetime of nucleotide sequences.

[0146] The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences discussed herein, or any derivative, fragment or derivatives thereof. If the sequence is complementary to a fragment thereof, then the sequence can be used as a probe to identify similar coding sequences in other organisms, etc.

[0147] Polynucleotides that are not 100% homologous to the sequences of the present invention, but which fall within the scope of the invention, can be obtained in various forms. Other variants of the sequences described herein can be obtained, for example, by probing DNA libraries made from a range of individuals, for example, individuals from different populations. In addition, other viral / bacterial or cellular homologs, particularly cellular homologs revealed in plant cells, can be obtained, and such homologs and fragments thereof will generally be able to selectively hybridize the sequences shown in the sequence listing herein. Such sequences Petition 870260073365, dated 07 / 23 / 2026, page 42 / 198 36 / 92 can be obtained by probing cDNA libraries or genomic DNA libraries of other plant species, and probing such libraries with probes comprising all or part of any of the sequences in the attached sequence listing under medium to high rigor conditions. Similar considerations are applied to obtain species homologs and allelic variants of the polypeptide or nucleotide sequences of the invention.

[0148] Variants and strain homologs can also be obtained using degenerate PGR which will use primers designed for target sequences in the variants and homologs that encode amino acid sequences conserved in the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning amino acid sequences from various variants / homologs. Sequence alignments can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used.

[0149] The primers used in degenerate PGR will contain one or more degenerate positions and will be used under lower rigor conditions than those used to clone sequences with single sequence primers against known sequences.

[0150] Alternatively, such polynucleotides can be obtained by directed mutagenesis of characterized sequences. This may be useful in cases where, for example, silent codon sequence changes are required to optimize codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired to introduce restriction polypeptide recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides. Petition 870260073365, dated 07 / 23 / 2026, page 43 / 198 37 / 92

[0151] Polynucleotides (nucleotide sequences) of the invention can be used to produce a primer, for example, a PGR primer, a primer for an alternative amplification reaction, a probe, for example, identified with a revealing label by conventional means using radioactive or non-radioactive identifications, or the polynucleotides can be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example, at least 25, 30 or 40 nucleotides in length, and are also covered by the term polynucleotides of the invention, as used herein.

[0152] Polynucleotides such as DNA polynucleotides and probes according to the invention can be produced recombinantly, synthetically, or by any means available to those skilled in the art. They can also be cloned by standard techniques.

[0153] In general, primers will be produced synthetically, involving a step-by-step fabrication of the desired nucleic acid sequence, one nucleotide at a time. Techniques to accomplish the same using automated techniques are readily available in the art.

[0154] Longer polynucleotides will generally be produced using recombinant media, for example, using PGR (polymerase chain reaction) cloning techniques. This will involve creating a primer pair (e.g., of about 15 to 30 nucleotides) flanking a region of the pyropheophytinase sequence to be cloned, bringing the primers into contact with mRNA or cDNA obtained from a plant cell, performing a polymerase chain reaction under conditions that bring about the enlargement of the desired region, isolating the enlarged fragment (e.g., by purifying the reaction mixture on a gel). Petition 870260073365, dated 07 / 23 / 2026, page 44 / 198 38 / 92 agarose) and recovering the enlarged DNA. Primers can be designed to contain suitable restriction enzyme recognition sites so that the enlarged DNA can be cloned into a suitable cloning vector. Enzyme Formulation and Dosage

[0155] Enzymes used in the methods of the invention may be formulated or modified, for example, chemically modified, to enhance oil solubility, stability, activity, or immobilization. For example, enzymes used in the methods of the invention may be formulated to be amphipathic or more lipophilic. For example, enzymes used in the methods of the invention may be encapsulated, for example, in liposomes or gels, for example, alginate hydrogels or alginate beads or equivalents. Enzymes used in the methods of the invention may be formulated in micellar systems, for example, a ternary micellar system (TMS) or reverse micellar system (RMS) medium. Enzymes used in the methods of the invention may be formulated as described in Yi (2002) J. of Molecular Catalysis B: Enzymatic, Volume 19, pages 319 to 325.

[0156] The enzymatic reactions of the methods of the invention, for example, the step of contacting the oil with an enzyme that performs chlorophyll hydrolysis or a chlorophyll derivative, can be carried out in one reaction vessel or multiple vessels. In one aspect, the enzymatic reactions of the methods of the invention are carried out in a vegetable oil or plant refining unit.

[0157] The method of the invention can be practiced with immobilized enzymes, for example, an immobilized chlorophyllase, pheophytinase and / or pyropheophytinase. The enzyme can be immobilized on any organic or inorganic support. Exemplary inorganic supports include alumina, celite, Dowex-1 in chloride form, glass beads and silica gel. Supports Petition 870260073365, dated 07 / 23 / 2026, page 45 / 198 39 / 92 Exemplary organic materials include DEAE-cellulose, alginate hydrogels or alginate beads or equivalents. In several aspects of the invention, enzyme immobilization can be optimized by physical adsorption onto the inorganic support. Enzymes used to practice the invention can be immobilized on different media, including water, Tris-HCl buffer solution and a ternary micellar system containing Tris-HCl buffer solution, hexane and surfactant. The enzyme can be immobilized on any type of substrate, for example, filters, fibers, columns, beads, colloids, gels, hydrogels, meshes and the like.

[0158] The enzyme can be measured in the oil in any suitable quantity. For example, the enzyme can be measured in a range of about 0.001 to 10 U / g of the composition, preferably 0.01 to 1 U / g, for example, 0.01 to 0.1 U / g of the oil. One unit is defined as the amount of enzyme that hydrolyzes 1 pmol of substrate (e.g., chlorophyll, pheophytin and / or pyropheophytin) per minute at 40°C, for example, under assay conditions as described in J. Biol. Chem. (1961) 236: 2544 to 2547. Phospholipid Content

[0159] In the process of the present invention, the enzyme comes into contact with the oil in the presence of at least 0.1% by weight of phospholipid. For example, the phospholipid content of the oil may be at least 0.1% by weight, for example, based on the total weight of the oil composition, for at least part of a time during which the enzyme is incubated with the oil (for example, at least once when the enzyme is added to the oil).

[0160] In some embodiments, for example, where the enzyme is added during the degumming step, the phospholipid content of the oil may decrease during the time the enzyme is incubated with the oil. However, it is provided that the phospholipid content of the oil is 0.1% by weight or above for at least part of the incubation with the enzyme (e.g., at the beginning of the Petition 870260073365, dated 07 / 23 / 2026, p. 46 / 198 40 / 92 incubation), the enzyme must be equally active. Therefore, in some embodiments, the phospholipid content of the oil may be less than 0.1% by weight during part of the incubation period with the enzyme.

[0161] Phospholipid is typically present as a natural component of a crude plant oil, although in some embodiments, the phospholipid may be added to the oil to be treated with the enzyme. Phospholipids commonly disclosed in crude plant oils include phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA). In preferred embodiments, the phospholipid comprises one or more of PC, PI, PE, PS, and PA. Phospholipids are typically present in crude oils in the form of lecithin, the main component being PC. Then in one embodiment, the phospholipid comprises lecithin. The term lecithin as used herein encompasses phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid.

[0162] The phospholipid content (e.g., PC, PI, PE, PS, PA and / or lecithin) of the oil is at least 0.1% by weight during contact with the enzyme. In particular embodiments, the phospholipid content is at least 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9% or at least 3.0% by weight, for example, based on the total weight of the oil. Preferably, the phospholipid content is up to 5.0%, up to 4.0%, or up to 3.0% by weight. For example, the phospholipid content of the oil can be 0.1 to 5.0%, 0.1 to 4.0%, 0.1 to 3.0%, 0.3 to 5.0%, 0.3 to 4.0%, 0.3 to 3.0%, 0.5 to 5.0%, 0.5 to 4.0%, 0.5 to 3.0%, 1.0 to 5.0%, 1.0 to 4.0%, 1.0 to 3.0%, 2.0 to 5.0%, 2.0 to 4.0%, or 2.0 to 3.0% by weight, for example, based on the total weight of the oil.

[0163] The phospholipid content of plant oil varies according Petition 870260073365, dated 07 / 23 / 2026, p. 47 / 198 41 / 92 with the particular source and nature of the oil and the stage of the refining process. The phospholipid content of crude plant oils can be up to 5% by weight at the start of the process, but after the water degumming step, the phospholipid content typically drops to 1% by weight or below, for example, around 0.3% by weight. After an enzymatic degumming step (e.g., using a phospholipase) or a total degumming step (e.g., comprising an acid treatment / caustic neutralization) the phospholipid content can drop much further, for example, below 0.1% or even below 0.01% by weight based on the total weight of the oil. The typical phospholipid content in % by weight of some common oils is shown below: Table 4 Canola Rapeseed Soybean Crude Oil <2.5 <3.5 <4.0 Water-degummed oil <0.6 <0.8 <0.4 Acid-degummed oil <0.1 <0.2

[0164] The values ​​in the table above are taken from Bailey's Industrial Products of Petroleum and Fat (2005), 6th edition, ed. by Fereidoon Shahidi, John Wiley & Sons, and the phospholipid content of other oils is also described therein or is well known in the art. The phospholipid content of oils can be determined using standard methods. For example, phospholipid levels in oils can be determined as described in J. Amer. Oil. Chem. Soc. 58, 561 (1981). In one embodiment, phospholipid levels can be determined by thin-layer chromatography (TLC) analysis, for example, as described in documents WO 2006 / 008508 or WO 03 / 100044. Phospholipid levels in oil can also be determined by (a) AOCS Recommended Practice Ca 19 to 86 (reapproved in 2009), Phospholipids in Vegetable Oils Nephelometric Method or (b) AOCS Official Method Ca 20 to 99 (reapproved in 2009), Analysis for Phosphorus in Oil by Inductively Coupled Plasma Optical Emission Petition 870260073365, dated 07 / 23 / 2026, p. 48 / 198 42 / 92 Spectroscopy.

[0165] Thus, in a preferred embodiment, the enzyme is brought into contact with a crude plant oil (for example, an oil comprising at least 0.5%, at least 1.0%, or at least 2% by weight of phospholipid). In another embodiment, the enzyme is brought into contact with a water-degummed plant oil (for example, an oil comprising 0.1 to 1% by weight of phospholipid). Lysophospholipid content

[0166] In a preferred embodiment of the process, the enzyme is brought into contact with the oil at a time when the concentration of lysophospholipid in the oil is as low as possible. For example, the enzyme may be brought into contact with the oil in the presence of less than 0.2% by weight of lysophospholipid. By "in the presence of less than 0.2% by weight of lysophospholipid" it is meant that the lysophospholipid content in the oil is less than 0.2% by weight, for example, based on the total weight of the oil composition, for at least part of a time during which the enzyme is incubated with the oil (for example, at least at one time when the enzyme is added to the oil). The lysophospholipid content in the oil may be any value below 0.2% by weight, including zero.

[0167] In some embodiments, for example, where the enzyme is added during a degumming step, the lysophospholipid content of the oil may increase during the time the enzyme is incubated with the oil. This is particularly the case where the process comprises an enzymatically degumming step using an enzyme that generates lysophospholipids. Lysophospholipids are typically produced during oil processing by cleaving an acyl (fatty acid) chain from phospholipids, leaving a single acyl chain, a phosphate group, optionally a head group, and a free alcohol attached to the glyceryl chemical portion. The Petition 870260073365, dated 07 / 23 / 2026, p. 49 / 198 43 / 92 Enzymes used in degumming, such as phospholipases (in particular phospholipase A1 and A2) and acyl transferases, can generate lysophospholipids in the oil. In embodiments where the process comprises an enzymatic degumming step using an enzyme that generates lysophospholipids, the enzyme that hydrolyzes chlorophyll or a chlorophyll derivative is preferably brought into contact with the oil before the enzymatic degumming step.

[0168] In one embodiment, a lysophospholipase can be used in combination with a phospholipase or acyltransferase in the degumming step. Lysophospholipases (EC 3.1.1.5) are enzymes that can hydrolyze lysophospholipids to release fatty acid. The use of a lysophospholipase can help reduce the production of lysophospholipids in the oil during the degumming step, for example, to keep the lysophospholipid content of the oil below about 0.2% by weight. Suitable lysophospholipases are disclosed, for example, in Masuda et al., Eur. J. Biochem., 202,783 to 787 (1991); WO 98 / 31790; WO 01 / 27251 and WO 2008 / 040465.

[0169] Phospholipase C is another enzyme that can be used in degumming. Phospholipase C cleaves phospholipids between the glyceryl and phosphate chemical moieties, leaving diacylglycerol, and a phosphate group (attached to a head group, if present). Thus, in contrast to phospholipase A1 and A2, phospholipase C does not produce lysophospholipids. In embodiments where the process comprises an enzymatic degumming step using an enzyme that does not produce lysophospholipids (e.g., phospholipase C), the enzyme that hydrolyzes chlorophyll or a chlorophyll derivative can be brought into contact with the oil either before or during the enzymatic degumming step.

[0170] In particular embodiments, the lysophospholipid content of the oil is less than 0.2%, less than 0.15%, less than 0.1%, or less than 0.05% by weight, based on the total weight of the oil. In general, lysophospholipid concentrations that are as low as possible are desirable. Petition 870260073365, dated 07 / 23 / 2026, page 50 / 198 44 / 92

[0171] Lysophospholipids that may be present in the oil include lysophosphatidylcholine (LPC), lysophosphatidylinositol (LPI), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), and lysophosphatidic acid (LPA). It is particularly preferred that the level of LPC and LPE in the oil be as low as possible. In preferred embodiments, the concentration of LPC and / or LPE is less than 0.2%, less than 0.15%, less than 0.1%, or less than 0.05% by weight, based on the total weight of the oil.

[0172] The lysophospholipid content of oils can be determined using standard methods, for example, as described above for phospholipids, including using HPLC or TLC analytical methods. Suitable methods are described in Recommended Practice by AOCS Ja 7 to 86 (re-approved in 2009), Phospholipids in Lecithin Concentrates by ThinLayer Chromatography or Journal of Chromatography A, 864 (1999) 179 to 182. ENZYME REACTION CONDITIONS

[0173] In general, the oil can be incubated (or mixed by addition) with the enzyme between about 5°C and about 100°C, more preferably between 10°C and about 90°C, more preferably between about 15°C and about 80°C, more preferably between about 20°C and about 75°C.

[0174] At higher temperatures, pheophytin is decomposed into pyropheophytin, which is generally less preferred since some chlorophyllases are less active on pyropheophytin compared to pheophytin. Furthermore, the chlorophyllase degradation product of pyropheophytin, pyrophorebidin, is less water-soluble compared to pheophorbidin and thus more difficult to remove from the oil after processing. The enzymatic reaction rate increases at higher temperatures, but it is advantageous to keep the conversion of pheophytin to pyropheophytin to a minimum. Petition 870260073365, dated 07 / 23 / 2026, page 51 / 198 45 / 92

[0175] In view of the above, in particularly preferred embodiments the oil is incubated with the enzyme at below about 80°C, preferably below about 70°C, preferably at or below about 68°C, preferably at or below about 65°C, in order to reduce the amount of conversion to pyropheophytin. However, in order to maintain a good reaction rate, it is preferable to keep the oil temperature above 50°C during incubation with the enzyme. Consequently, preferred temperature ranges for incubating the enzyme with the oil include about 50°C to below about 70°C, about 50°C to about 65°C and about 55°C to about 65°C. Preferably, the enzyme is brought into contact with the oil at about 57°C to about 63°C, preferably about 58°C to about 62°C, for example, about 60°C.

[0176] Preferably, the oil temperature may be at the desired reaction temperature when the enzyme is added to it. The oil may be heated and / or cooled to the desired temperature before and / or during the addition of enzyme. Therefore, in one embodiment, it is conceived that an additional step of the process according to the present invention may be the cooling and / or heating of the oil.

[0177] Suitably, the reaction time (i.e., the period of time during which the enzyme is incubated with the oil), preferably with agitation, is for a period of time sufficient to allow the hydrolysis of chlorophyll and chlorophyll derivatives, for example, to form phytol and chlorophyllide, pheophorbide and / or pyropheophorbide. For example, the reaction time may be at least about 1 minute, more preferably at least about 5 minutes, most preferably at least about 10 minutes. In some embodiments the reaction time may be between about 15 minutes and about 6 hours, preferably between about 15 minutes and about 60 minutes, preferably about 30 to about 120 minutes. Petition 870260073365, dated 07 / 23 / 2026, p. 52 / 198 46 / 92 minutes. In some scenarios, reaction time can be up to 6 hours.

[0178] Preferably the process is carried out between about pH 4.0 and about pH 10.0, more preferably between about pH 5.0 and about pH 10.0, more preferably between about pH 6.0 and about pH 10.0, more preferably between about pH 5.0 and about pH 7.0, more preferably between about pH 5.0 and about pH 7.0, more preferably between about pH 6.5 and about pH 7.0, for example at about pH 7.0 (i.e., neutral pH). In one embodiment, preferably the process is carried out between about pH 5.5 and pH 6.0. In another embodiment, the process is carried out between about pH 6.0 and pH 6.8, for example, between about pH 6.3 and pH 6.5, preferably about pH 6.4.

[0179] Suitably, the water content of the oil when incubated (or mixed by addition) with the enzyme is between about 0.5 to about 5% water, more preferably between about 1 to about 3% and most preferably between about 1.5 and about 2% by weight. In specific embodiments, the water content may be, for example, 0.7% to 1.2%, for example, about 1% by weight; or 1.7% to 2.2%, for example, about 2% by weight.

[0180] When an immobilized enzyme is used, the water activity of the immobilized enzyme may suitably be in the range of about 0.2 to about 0.98, preferably between about 0.4 and about 0.9, more preferably between about 0.6 and about 0.8. Oil Separation

[0181] Following a step of enzymatic treatment using an enzyme according to the present invention, in one embodiment the treated liquid (e.g., oil) is separated with a suitable means such as a centrifugal separator and the processed oil is obtained. Upon completion of the Petition 870260073365, dated 07 / 23 / 2026, page 53 / 198 47 / 92 Enzyme treatment, if necessary, the processed oil may be additionally washed with water or organic or inorganic acid such as, for example, acetic acid, citric acid, phosphoric acid, succinic acid and the like, or with salt solutions. Removal of Chlorophyll and / or Chlorophyll Derivatives

[0182] The process of the present invention involving enzyme treatment typically reduces the level of chlorophyll and / or chlorophyll derivatives in the oil. For example, the process can reduce the concentration of chlorophyll, pheophytin and / or pyropheophytin by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%, compared to the concentration of chlorophyll, pheophytin and / or pyropheophytin (by weight) present in the oil before treatment. Thus, in particular embodiments, the concentration of chlorophyll and / or chlorophyll derivatives in the oil after treatment may be less than 100, less than 50, less than 30, less than 10, less than 5, less than 1, less than 0.5, less than 0.1 mg / kg or less than 0.02 mg / kg, based on the total weight of the oil. Additional Processing Steps

[0183] In a typical plant oil processing method, the oil is extracted with hexane, the crude vegetable oil is degummed, optionally caustic-neutralized, bleached using, for example, clay absorption with subsequent clay disposal, and deodorized to produce refined, bleached and deodorized oil or RBD (see Figure 31). The need for the degumming step depends on the phosphorus content and other factors. The process of the present invention can be used in conjunction with processes based on hexane extraction and / or enzyme-assisted oil extraction (see Journal of America. Oil Chemists' Society (2006), 83 (11), Petition 870260073365, dated 07 / 23 / 2026, page 54 / 198 48 / 92 973 to 979). In general, the invention process can be carried out using oil processing steps as described in Bailey's Industrial Oil and Fat Products (2005), 6th edition. Edited by Fereidoon Shahidi, John Wiley & Sons.

[0184] In embodiments of the present invention, an enzymatic reaction involving the application of an enzyme capable of hydrolyzing chlorophyll or a chlorophyll derivative is preferably carried out at specific stages in this process. In particular, according to the present invention, the enzyme is brought into contact with the oil in the presence of at least 0.1% by weight of phospholipid and preferably less than 0.2% by weight of lysophospholipid. Although the level of phospholipid and lysophospholipid in the oil at different stages of the process varies depending on the nature and source of the oil, it is generally preferable to bring the enzyme into contact with the oil at a stage in the process before phospholipid levels are substantially reduced and before lysophospholipid levels are elevated.

[0185] Preferred stages of the process for the use of the enzyme according to the present process are shown in Figure 31. In particular embodiments, the enzyme is preferably brought into contact with the oil before the degumming step. In another embodiment, the enzyme may be brought into contact with the oil during the water degumming step. The enzyme is typically brought into contact with the oil before degumming is completed (e.g., before a caustic neutralization step).

[0186] In some embodiments, the enzyme can be brought into contact with the oil after degumming with water (for example, the enzyme is added to oil degummed with water), provided that the enzymatic hydrolysis of chlorophyll and chlorophyll derivatives is carried out before a complete degumming step, for example, before the addition of acid and caustic neutralization. This is shown by a dashed line in Figure 31. Thus, the enzyme can Petition 870260073365, dated 07 / 23 / 2026, page 55 / 198 49 / 92 can be added after partial degumming of the oil, provided that at least 0.1% phospholipid by weight is still present. In general, however, it is preferable to add the enzyme at as high a phospholipid level as possible (e.g., preferably at least 0.5% or 1.0% by weight of phospholipid), and the use of the enzyme after partial degumming is generally less preferred.

[0187] Additional processing steps, after treatment with the enzyme, may assist in the removal of enzymatic hydrolysis products of chlorophyll and / or chlorophyll derivatives. For example, additional processing steps may remove chlorophyllide, pheophorbide, pyropheophorbide and / or phytol. Degumming

[0188] The degumming step in oil refining serves to separate phosphatides by adding water. The material precipitated by degumming is separated and further processed into lecithin mixtures. Commercial lecithins, such as soybean lecithin and sunflower lecithin, are semi-solid or very viscous materials. They consist of a mixture of polar lipids, primarily phospholipids such as phosphatidylcholine with a minor component of triglycerides. Thus, as used in this document, the term degumming means oil refining by removing phospholipids from the oil. In some embodiments, degumming may comprise a step converting phosphatides (such as lecithin and phospholipids) into hydratable phosphatides.

[0189] The invention process can be used with any degumming procedure, particularly in embodiments where the chlorophyll hydrolyzing enzyme or chlorophyll derivative is brought into contact with the oil before the degumming step. Thus, suitable degumming methods include water degumming, ALCON oil degumming (e.g., for soybeans), saffin degumming, super degumming, UF degumming, Petition 870260073365, dated 07 / 23 / 2026, page 56 / 198 50 / 92 TOP degumming, single-degumming, dry degumming and ENZYMAX™ degumming. See, for example, US Patents No. 6,355,693; 6,162,623; 6,103,505; 6,001,640; 5,558,781; 5,264,367; 5,558,781; 5,288,619; 5,264,367; 6,001,640; 6,376,689; WO 0229022; WO 98118912; and similar patents. Several degumming procedures incorporated by the methods of the invention are described in Bockisch, M. (1998), Fats and Oils Handbook, The Extraction of Vegetable Oils (Chapter 5), 345 to 445, AOCS Press, Champaign, Illinois, USA.

[0190] Degumming with water typically refers to a step in which the oil is incubated with water (e.g., 1 to 5% by weight) in order to remove phosphatides. Typically, degumming with water can be carried out at elevated temperatures, for example, at 50 to 90°C. The oil / water mixture can be stirred for, for example, 5 to 60 minutes to allow the separation of phosphatides in the water phase, which is then removed from the oil.

[0191] Acid degumming can also be performed. For example, oil can be brought into contact with acid (e.g., 0.1 to 0.5% of a 50% citric or malic acid solution) at 60 to 70°C, mixed, brought into contact with 1 to 5% water and cooled to 25 to 45°C.

[0192] Additional degumming procedures suitable for use with the process of the present invention are described in WO 2006 / 008508. In one embodiment, the process comprises placing chlorophyll hydrolyzing enzyme or chlorophyll derivative in contact with the oil and subsequently performing an enzymatic degumming step using an acyltransferase, as described in WO 2006 / 008508. Acyltransferases suitable for use in the process are also described in WO 2004 / 064537, WO 2004 / 064987 and WO 2009 / 024736. Any enzyme having acyltransferase activity (generally classified as EC2.3.1) can be used, particularly enzymes comprising the GDSX amino acid sequence motif, in Petition 870260073365, dated 07 / 23 / 2026, page 57 / 198 51 / 92 that X is one or more of the following amino acid residues: L, A, V, I, F, Y, H, Q, T, N, M, or S. In one embodiment, acyltransferase is a mature lipid acyltransferase from mutant Aeromonas salmonicida (CSCAT) with an Asn80Asp mutation, for example, an acyltransferase comprising the amino acid sequence of SEQ ID NO: 23 after undergoing post-translational modification (see Figure 32), or an enzyme that has at least 80% sequence identity to it.

[0193] In another embodiment, the process comprises the degumming step using a phospholipase. Any enzyme having, for example, phospholipase A1 (EC3.1.1.32) or phospholipase A2 (EC3.1.1.4) activity can be used, for example, Lecitase Ultra® or pancreatic phospholipase A2 (Novozymes, Denmark). In one embodiment, the process comprises placing the chlorophyll hydrolyzing enzyme or chlorophyll derivative in contact with the oil and subsequently performing an enzymatic degumming step using a phospholipase, for example, using a degumming step as described in US documents 5,264,367, EP 0622446, WO 00 / 32758 or Clausen (2001) Enzymatic oil degumming by a novel microbial phospholipase, Eur. J. Lipid Sci. Technol. 103:333 to 340.

[0194] In embodiments where the degumming step is performed simultaneously with the step of hydrolyzing chlorophyll or chlorophyll derivative, the degumming process preferably does not produce lysophospholipids. For example, in these embodiments the degumming step may be a water degumming step. In another such embodiment, an enzymatic degumming step using an enzyme such as phospholipase C (IUB 3.1.4.1) may be used. Polypeptides that have phospholipase C activity that can be used in a degumming step are disclosed, for example, in documents WO2008143679, WO2007092314, WO2007055735, WO2006009676 and WO03089620. A phospholipase C suitable for use in Petition 870260073365, dated 07 / 23 / 2026, page 58 / 198 52 / 92 The present invention is Purifine®, available from Verenium Corporation, Cambridge, MA. Acid Treatment / Caustic Neutralization

[0195] In some embodiments, an acid treatment / caustic neutralization step may be performed in order to further reduce phospholipid levels in the oil after water degumming. In another embodiment, a single degumming step comprising acid treatment / caustic neutralization may be performed. Such methods are typically referred to as total degumming or alkali refining.

[0196] It has been found that an acid treatment / caustic neutralization step is particularly effective in removing products of enzymatic hydrolysis of chlorophyll, for example, chlorophyllide, pheophorbide and pyropheophorbide. Thus, this step can be performed at any stage in the process after the enzyme treatment step. For example, such a step may comprise the addition of an acid, such as phosphoric acid, followed by neutralization with a sodium hydroxide or alkali. Following an acid treatment / caustic neutralization, compounds such as chlorophyllide, pheophorbide and pyropheophorbide are extracted from the oil in an aqueous phase.

[0197] In such methods, the oil is typically first brought into contact with 0.05 to 0.5% by weight of concentrated phosphoric acid, for example, at a temperature of 50 to 90°C, and mixed to help precipitate the phosphatides. The contact time may be, for example, from 10 seconds to 30 minutes. Subsequently, an aqueous solution of an alkali (for example, 1 to 20% aqueous sodium hydroxide) is added, for example, at a temperature of 50 to 90°C, followed by incubation and mixing for 10 seconds to 30 minutes. The oil may then be heated to about 90°C and the aqueous soap phase separated from the oil by centrifugation.

[0198] Optionally, additional washing steps with, for Petition 870260073365, dated 07 / 23 / 2026, p. 59 / 198 53 / 92 For example, sodium hydroxide or water can also be used. Removal of Chlorophyllide, Pheophorbidum and Pyropheophorbidum

[0199] Thus, the method of the present invention may optionally include a step to remove phytol-free derivatives of chlorophyll such as chlorophyllide, pheophorbide, and pyropheophorbide. Such products may be present in the composition due to the hydrolysis of chlorophyll or a chlorophyll derivative by the enzyme of the invention, or may be present naturally as a contaminant or as an undesirable component in a processed product. Pyropheophorbide may also be present in the composition due to the breakdown of pheophorbide, which may itself be produced by the activity of an enzyme that has pheophytinase activity on pheophytin, or pheophorbide may be formed from chlorophyllide following the action of chlorophyllase on chlorophyll (see Figure 1).The processing conditions used in oil refining, particularly heat, can favor the formation of pyropheophorbides as a dominant component, for example, by favoring the conversion of pheophytin to pyropheophytin, which is subsequently hydrolyzed to pyropheophorbides.

[0200] In one embodiment, the process of the present invention reduces the level of chlorophyllide, pheophorbide and / or pyropheophorbide in the oil, compared to one or both levels before and after enzyme treatment. Thus, in some embodiments, the concentration of chlorophyllide, pheophorbide and / or pyropheophorbide may increase after enzyme treatment. Typically, the process involves a step to remove chlorophyllide, pheophorbide and / or pyropheophorbide so that the concentration of such products is lower than after enzyme treatment. Preferably, the chlorophyllide, pheophorbide and / or pyropheophorbide produced by this enzymatic step is removed from the oil, so that the final level of these products in the oil is lower than before enzyme treatment. Petition 870260073365, dated 07 / 23 / 2026, page 60 / 198 54 / 92

[0201] For example, the process can reduce the concentration of chlorophyllide, pheophorbide and / or pyropheophorbide by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%, compared to the concentration of chlorophyllide, pheophorbide and / or pyropheophorbide (by weight) present in the oil before the step of removing chlorophyllide, pheophorbide and / or pyropheophorbide, that is, before or after enzyme treatment. Thus, in particular embodiments, the concentration of chlorophyllide, pheophorbide and / or pyropheophorbide in the oil after the removal step may be less than 100, less than 50, less than 30, less than 10, less than 5, less than 1, less than 0.5, less than 0.1 mg / kg, or less than 0.02 mg / kg, based on the total weight of the composition (e.g., a vegetable oil).

[0202] It is an advantage of the present process that reaction products, such as chlorophyllide, pheophorbide and / or pyropheophorbide, can be simply and easily removed from the oil through a single step, such as acid treatment / caustic neutralization. Thus, in preferred embodiments, chlorophyll and chlorophyll derivatives can be substantially removed from the oil without the need for additional processing steps such as deodorizing and silica and / or clay treatment (as indicated by the dotted boxes shown in Figure 31). Clay Treatment

[0203] It is particularly preferable that the process does not include a clay treatment step. Avoiding the use of clay is advantageous for the reasons described above, in particular the reduction in cost, reduced oil losses through adherence to clay and greater retention of useful compounds such as carotenoids and tocopherol.

[0204] In some realizations, the process may be Petition 870260073365, dated 07 / 23 / 2026, page 61 / 198 55 / 92 performed with no clay treatment step and no deodorizing step, resulting in a higher concentration of such beneficial compounds in the refined oil, compared to a process involving clay treatment. Silica Treatment

[0205] Although not always required, in some embodiments, the process may comprise a silica treatment step, preferably subsequent to enzyme treatment. For example, the method may comprise the use of reduced-adsorbance or adsorbence-free silica refining processes and devices, which are known in the art, for example, with the use of TriSyl Silica Refining Processes (Grace Davison, Columbia, MD), or SORBSIL R™ silicas (INEOS Silicas, Joliet, IL).

[0206] The silica treatment step can be used to remove any remaining chlorophyllide, pheophorbide and / or pyropheophorbide or other polar components in the oil. For example, in some embodiments, a silica treatment step can be used as an alternative to an acid treatment / caustic neutralization step (total degumming or alkali refining).

[0207] In one embodiment, the process comprises a two-stage silica treatment, for example, comprising two silica treatment steps separated by a separation step in which the silica is removed, for example, a filtering step. The silica treatment can be carried out at elevated temperatures, for example, above about 30°C, more preferably about 50 to 150°C, about 70 to 110°C, about 80 to 100°C or about 85 to 95°C, most preferably about 90°C. Deodorization

[0208] In some realizations, the process may include Petition 870260073365, dated 07 / 23 / 2026, page 62 / 198 56 / 92 a deodorizing step, typically as the final refining step in the process. In one embodiment, deodorization refers to the steam distillation of the oil, which typically removes volatile taste and odor compounds, tocopherol, sterols, stanols, carotenoids, and other nutrients. Typically, the oil is heated to 220 to 260°C under low pressure (e.g., 0.1 to 1 kPa) to exclude air. Steam (e.g., 1 to 3% by weight) is blown through the oil to remove volatile compounds, for example, for 15 to 120 minutes. The aqueous distillate may be collected.

[0209] In another embodiment, deodorization can be performed using an inert gas (e.g., nitrogen) instead of steam. Thus, the deodorizing step may comprise bubble refining or spraying with an inert gas (e.g., nitrogen), for example, as described by AV Tsiadi et al. in Nitrogen bubble refining of sunflower oil in shallow pools, Journal of the American Oil Chemists' Society (2001), Volume 78 (4), pages 381 to 385. The gaseous phase that has passed through the oil can be collected and optionally condensed, and / or have volatile compounds extracted from it in an aqueous phase.

[0210] In some embodiments, the process of the present invention is carried out with no clay treatment, but comprising a deodorizing step. Useful compounds (e.g., carotenoids, sterols, stanols, and tocopherol) can be at least partially extracted from the oil in a distillate (e.g., an aqueous or nitrogenous distillate) obtained from the deodorizing step. This distillate provides a valuable source of compounds such as carotenoids and tocopherol, which can be at least partially lost by carryover in a process comprising clay treatment.

[0211] The loss of tocopherol during bleaching depends on the bleaching conditions and the type of clay applied, but 20 to 40% of Petition 870260073365, dated 07 / 23 / 2026, page 63 / 198 57 / 92 loss of tocopherol in the bleaching step has been reported (K. Boki, M. Kubo, T. Wada, and T. Tamura, ibid., 69, 323 (1992)). During soybean oil processing, a 13% loss of tocopherol in the bleaching step has been reported (S. Ramamurthi, A.R. McCurdy, and R.T. Tyler, in S.S. Koseoglu, K.C. Rhee, and R.F. Wilson, eds., Proc. World Conf. Oilseed Edible Oils Process, volume 1, AOCS Press, Champaign, Illinois, 1998, pages 130-134).

[0212] Carotenoids can be removed from oil during deodorization in both clay-treated and non-clay-treated oil. Typically, the removal of colored carotenoids is controlled in order to produce an oil that has a predetermined color within a specific range of values. The level of carotenoids and other volatile compounds in the refined oil can be varied by modifying the deodorizing step. For example, in an embodiment where it is desirable to retain a higher concentration of carotenoids in the oil, the deodorizing step can be performed at a lower temperature (e.g., using steam at 200°C or below). In such embodiments, it is particularly preferable to avoid a clay treatment step, as this will result in a higher concentration of carotenoids in the refined oil. Treatments with Additional Enzymes

[0213] In additional aspects, the processes of the invention further comprise the use of lipid acyltransferases, phospholipases, proteases, phosphatases, phytases, xylanases, amylases (e.g., α-amylases), glucanases, polygalacturonases, galactolipases, cellulases, hemicellulases, pectinases and other plant cell wall degradation enzymes, as well as mixed enzyme preparations and cell lysates. In alternative aspects, the processes of the invention can be carried out in conjunction with other processes, for example, enzymatic treatments, for example, with carbohydrases, including cellulase, hemicellulase and other side degradation activities, or chemical processes, for example, hexane extraction from soybean oil. In one embodiment, the Petition 870260073365, dated 07 / 23 / 2026, page 64 / 198 58 / 92 The method of the present invention can be practiced in combination with a method as defined in document WO 2006031699.

[0214] The invention will now be further illustrated with reference to the following non-limiting examples. Example 1 Cloning and Expression of a Chlorophyllase from Triticum aestivum (Wheat) in Bacillus subtilis

[0215] A nucleotide sequence (SEQ ID No. 3) encoding a wheat chlorophyllase (SEQ. ID No. 2, hereinafter in this document wheat chlorophyllase) was expressed in Bacillus subtilis with the signal peptide of an alkaline protease from B. subtilis (aprE) (see Figure 17). For optimal expression in Bacillus, an optimized codon gene construct (TRI_CHL) was ordered from GenScript (GenScript Corporation, Piscataway, NJ 08854, USA).

[0216] The TRI_CHL construct contains 20 nucleotides with a BssHII restriction site upstream of the wheat chlase coding region to allow fusion to the aprE signal sequence and a PacI restriction site following the coding region for cloning into the pBNppt bacillus expression vector.

[0217] The TRI_CHL construct was digested with BssHII and PacI and ligated with T4 DNA ligase in pBNppt digested by BssHII and PacI.

[0218] The linkage mixture was transformed into TOP10 E. coli cells. The sequence of the Pac and BssHII insert containing the TRI_CHL gene was confirmed by DNA sequencing (DNA Technology A / S, Risskov, Denmark) and one of the correct plasmid clones was designated pBN-TRI_CHL (Figure 18). pBN-TRI_CHL was transformed into B. subtilis strain BG 6002, a derivative of A 2200, as described in document WO 2003 / 099843. Petition 870260073365, dated 07 / 23 / 2026, page 65 / 198 59 / 92

[0219] A neomycin-resistant transformant (neoR) was selected and used for the expression of wheat chlase. Example 2 Cloning and Expression of a Chlorophyllase from Chlamydomonas reinhardtii (Green Algae) in Bacillus subtilis

[0220] A nucleotide sequence (SEQ ID No. 5) encoding a Chlamydomonas chloryphyllase (SEQ. ID No. 4, hereinafter referred to in this document as chlamy chlase) was expressed in Bacillus subtilis with the signal peptide of an alkaline protease from B. subtilis (aprE) (see Figures 19 and 20). For optimal expression in Bacillus, an optimized codon gene construct (CHL_CHL) was ordered from GenScript (GenScript Corporation, Piscataway, NJ 08854, USA).

[0221] The CHL_CHL construct contains 20 nucleotides with a BssHII restriction site upstream of the chlamy chlase coding region to allow fusion with the aprE signal sequence and a PacI restriction site following the coding region for cloning into the pBNppt bacillus expression vector.

[0222] The CHL_CHL construct was digested with BssHII and Pa and ligated with T4 DNA ligase in PBNppt digested by BssHII and PacI.

[0223] The linkage mixture was transformed into TOP10 E. coli cells. The sequence of the BssHII and Pac insert containing the CHL_CHL gene was confirmed by DNA sequencing (DNA Technology A / S. Risskov, Denmark) and one of the correct plasmid clones was designated pBN-CHL_CHL (Figure 20). pBN-CHL_CHL was transformed into B. subtilis strain BG 6002, a derivative of AK 2200, as described in document WO 2003 / 099843.

[0224] A neomycin-resistant transformant (neoR) was selected and used for the expression of chlamy chlase. Petition 870260073365, dated 07 / 23 / 2026, page 66 / 198 60 / 92 Example 3 Effect of Surfactants on Chlorophylase Activity in Plant Oil

[0225] The activity of an Arabidopsis thaliana chlorophyllase with the sequence SEQ ID NO: 1 was tested in refined rapeseed oil with the addition of different surfactants, including soy lecithin. Samples 1 to 6 were prepared comprising the components defined in Table 5: Table 5 1 2 3 4 5 6 Refined rapeseed oil g 10 10 10 10 10 10 Soy lecithin g 0.2 0.2 Sorbitan monooleate g 0.2 0.2 Sorbitan trioleate g 0.2 0.2 0.5 mg / ml of chlorophyll in acetone ml 250 250 250 250 250 250 Chlorophyllase in Buffer* ml 0 0.25 0 0.25 0 0.25 Buffer* ml 0.3 0.05 0.3 0.05 0.3 0.05 % Water % 3.00 3.00 3.00 3.00 3.00 3.00 *Buffer: 0.24% Triton X100, 50 mM KCl, 100 mM Phosphate, pH=7.0

[0226] Refined rapeseed oil and surfactant were heated to 45°C with stirring. Chlorophyll, buffer and chlorophyllase were added. Samples were incubated with stirring for 180 minutes and 1 ml of sample was withdrawn and centrifuged for 3 minutes at 3,000 fcr.

[0227] The oil phase was measured by fluorescence spectroscopy (excitation at 410 nm, emission at 672 nm) and the amount of chlorophyll was quantified (Table 6) from a calibration curve produced from the measurement of refined rapeseed oil to which a known concentration of chlorophyll was added. Petition 870260073365, dated 07 / 23 / 2026, page 67 / 198 61 / 92 Table 6 Sample ppm of Chlorophyll 1 7.27 2 2.78 3 7.77 4 3.83 5 7.50 6 6.25

[0228] The results in Table 6 clearly indicate that different surfactants had a strong impact on chlorophyllase activity. Lecithin has a strong positive effect on chlorophyllase activity and thus on the reduction of chlorophyll in oil. Sorbitan monooleate also has a positive effect on chlorophyllase activity, but chlorophyllase activity is very modest when sorbitan trioleate is added to the oil.

[0229] As can be seen in Figure 21, chlorophyllase has maximum efficacy in combination with lecithin (sample 1). In contrast, chlorophyllase activity is low in combination with sorbitan trioleate (sample 6). It is also observed that sample 2 is more brownish than the other. This can be explained by the fact that some of the phospholipids, such as phosphatidic acid in lecithin, effectively complex with magnesium and thus convert chlorophyll into pheophytin (without magnesium). The results suggest that phospholipids (such as those present in lecithin) promote the hydrolysis of chlorophyll and chlorophyll derivatives through chlorophyllase. Example 4 Effect of Refining on Chlorophyllase Activity in Plant Oil

[0230] As shown in Example 3, surfactants influence chlorophyllase activity in refined oil. At different stages in the oil refining process, the amount of surfactant (particularly lecithin) can vary, thus influencing chlorophyllase activity.

[0231] In the following example, chlorophyllase activity was tested. Petition 870260073365, dated 07 / 23 / 2026, page 68 / 198 62 / 92 in a refined oil and in a combination of refined oil and crude soybean oil according to the recipe in Table 7: Table 7 1 2 3 4 5 6 7 8 Refined oil 10 10 10 10 Refined oil: Crude soybean oil 1:1 g 10 10 10 10 Chlorophyllase (Arabidopsis) ml 0 0 0.25 0.25 0 0 0.25 0.25 Chlorophyll ml 0.2 0 0.2 0 0.2 0 0.2 0 Extra Water ml 0.3 0.3 0.05 0.05 0.3 0.3 0.05 0.05 % water 3 3 3 3 3 3 3 3

[0232] The oil was heated to 40°C. Chlorophyll, water, and enzyme were added, and the sample was homogenized with high-shear mixing for 20 seconds and incubated at 40°C with magnetic stirring. After 90 minutes, the samples were heated to 97°C for 10 minutes and centrifuged at 1,780 fcr for 3 minutes.

[0233] The samples were visually evaluated, as shown in Figure 22. It is observed that sample 3 treated with refined oil chlorophyllase is not very different from sample 1, in which no chlorophyllase was added. If the enzyme had hydrolyzed chlorophyll, the chlorophyllide of the reaction product would appear with a green color in the lower water phase.

[0234] In sample 7 (a 1:1 mixture of refined oil: crude soybean oil treated with chlorophyllase) it is very clear that the green color enters the water phase and the water phase is very different from sample 5 in which no chlorophyllase was added.

[0235] The results show that chlorophyllase is only active in the hydrolysis of chlorophyll in oil samples containing crude soybean oil. This suggests that surfactants present in crude soybean oil facilitated the chlorophyllase reaction. Crude soybean oil contains a high level of surfactant in the form of 2a Petition 870260073365, dated 07 / 23 / 2026, page 69 / 198 63 / 92 3% lecithin (mainly phospholipids). In refined oil there are almost no surfactants and therefore no chlorophyllase activity is observed. Example 5 Effect of Lecithin and Acyltransferase on Chlorophylase Activity in Oil

[0236] Examples 3 and 4 suggest that surfactants such as lecithin (which comprise phospholipids) are important for chlorophyllase activity. Lecithin is present at varying levels in some crude plant oils. It is a natural constituent of crude plant oils such as soybean oil and rapeseed oil, but during the oil refining process, lecithin is typically removed from the oil by a degumming process. During the degumming step, lecithin can be removed enzymatically by enzymes such as phospholipases. If chlorophyllase activity is dependent on the presence of lecithin, the modification of lecithin by enzymes will impact chlorophyllase activity. It is therefore important for effective chlorophyll removal that chlorophyllase be used in the presence of a minimum level of lecithin.This can be influenced by the level of lecithin naturally present in the particular oil, a point during the refining process at which chlorophyllase is applied, and the nature of the degumming step.

[0237] In the following example, a crude oil was degummed with water without and with a lipid acyltransferase (LysoMax Oil® from Danisco A / S). LysoMax Oil® is a mature lipid acyltransferase from Aeromonas salmonicida (GCAT) with an AsnXOAsp mutation, comprising the amino acid sequence SEQ ID NO: 23 (see Figure 32). This enzyme is known to be very active on phospholipids during the formation of lysophospholipids. The isolated gum phase (lecithin or modified lecithin from LysoMax Oil®) from the water degumming was isolated and added to a refined oil in different amounts and then combined with 3% water and chlorophyllase in order to investigate the effect of the amount of lecithin and the type of lecithin. Petition 870260073365, dated 07 / 23 / 2026, page 70 / 198 64 / 92

[0238] Degumming with water was carried out according to the recipe in Table 8 shown: Table 8 AB Crude rapeseed oil g 150 150 LysoMax Oil® 100 U / ml* ml 0 0.2 Water ml 2.250 2.050 Enzyme oil (LysoMax Oil®) U / g 0.00 0.13 % water 1.50 1.50 * Lipid acyltransferase activity can be determined as described in document WO 2004 / 064987.

[0239] Crude oil was heated to 55°C. Water and enzyme were added and mixed with high shear mixing for 20 seconds followed by incubation with magnetic stirring. After 30 minutes of incubation, the samples were heated to 97°C for 10 minutes and centrifuged at 1,780 fcr for 3 minutes. The oil phase and the gum phase for the two experiments were isolated. The gum phases were dried in a rotary evaporator.

[0240] A Triticum chlorophyllase (see Example 1) was tested in a water degumming (WDG) process using oil and dry gum phase according to the formulas in Table 9. The oil and dry gum were heated to 55°C with stirring. Water and chlorophyllase were added. Samples were incubated at 65°C for 4 hours. The enzyme was inactivated by heating to 97°C for 10 minutes followed by centrifugation at 1,780 fcr for 3 minutes. Table 9 1 2 3 4 5 6 7 8 9 10 Degummed oil with water without A g 10 10 10 10 10 10 10 10 10 Raw rapeseed oil (Oct. 2009) g 10 Petition 870260073365, dated 07 / 23 / 2026, p. 71 / 198 65 / 92 1 2 3 4 5 6 7 8 9 10 Lecithin A g 0.02 0 0.05 0.1 0.2 Lecithin B (enzyme-modified) g 0.02 0.05 0.1 0.2 water ml 0.338 0.338 0.338 0.338 0.338 0.338 0.338 0.338 0.338 0.338 TRICHL CoRe 20 12.5 U / ml ml 0.012 0.012 0.012 0.012 0.012 0.012 0.012 0.012 0.012 0.012 Chlorophylase U / g of oil 0.015 0.015 0.015 0.015 0.015 0.015 0.015 0.015 0.015 0.015 % water 3.50 3.50 3.50 3.50 3.50 3.50 3.50 3.50 3.50 3.50

[0241] The oil phases were isolated and analyzed by HPLC with fluorescence detection. The RFU fluorescence signal was calculated based on the same amount of oil with the results in Table 10 and Figures 23 to 26. Table 10 % Gum A % Gum B Oil Phaeophorbide Pyropheophorbide Phaeophytin A Pyropheophytin 0.2 0 WDG without A 12.5 1.6 1.8 1.8 0 0.2 WDG without A 6.7 1.0 7.6 2.5 0.5 0 WDG without A 12.1 1.5 1.7 1.8 0 0.5 WDG without A 6.1 1.0 8.0 2.6 1 0 WDG without A 11.5 1.8 1.3 1.8 0 1 WDG without A 5.5 1.0 8.5 2.7 2 0 WDG without A 11.0 1.7 1.3 1.7 0 2 WDG without A 3.7 1.0 10.0 2.7 WDG Oil 0 WDG without A 11.0 1.5 2.9 2.3 Crude Oil 0 Crude Oil 10.5 13 0.6 0.9 WDG = water-degummed

[0242] For comparison, rapeseed oil without chlorophyllase treatment was analyzed with the following results: Table 11 Oil Rel. RFU Phaeophorbide Rel. RFU Pyropheophorbid ' Rel. RFU Feophytin Petition 870260073365, dated 07 / 23 / 2026, page 72 / 198 66 / 92 Oil Rel. RFU Phaeophorbide Rel. RFU Pyropheophorbid ' Rel. RFU Phaeophytin A Rel. RFU Pyropheophytin Rapeseed seed oil 4.5 0.9 11.9 2.4

[0243] The results in Table 10 clearly indicate an effect of lecithin and enzyme-modified lecithin on chlorophyllase activity. In the crude oil sample, chlorophyllase is clearly more active than in the water-degummed oil (WDG) sample. It is also observed that the addition of lecithin to the water-degummed oil increases chlorophyllase activity, and there is a dosage response from 0.2 to 2% lecithin added. The water-degummed oil contains approximately 0.3% lecithin, which is consistent with the fact that chlorophyllase is somehow active in the water-degummed oil without the addition of extra lecithin. As shown in Example 4, chlorophyllase activity is very slow in refined rapeseed oil, so the results suggest that the remaining lecithin in the water-degummed oil has a clear impact on chlorophyllase activity.

[0244] The addition of LysoMax Oil® modified lecithin (lipid acyl transferase from Danisco A / S) to water-degummed oil has a strong impact on chlorophyllase activity. Even a low level of enzyme-modified lecithin (0.2%) has a strong negative impact on chlorophyllase enzyme activity, and with the addition of 2% enzyme-modified lecithin, chlorophyllase activity is almost completely interrupted.

[0245] When lysolecithin was added to chlorophyllase in an aqueous system comprising Triton X100 as a surfactant, no reduction in chlorophyllase activity was observed (results not shown). This aqueous system differs markedly in terms of physical properties from an oily system containing only 3% water. The results suggest that lysophospholipids form different mesomorphic phases and thus Petition 870260073365, dated 07 / 23 / 2026, page 73 / 198 67 / 92 prevent the interaction between the substrate (e.g., pheophthin) and the enzyme (chlorophyllase). Example 6 Effect of Chlorophyllase and Phospholipases on Degumming with Rapeseed Oil Water

[0246] The results in Example 5 indicate that enzyme-modified lecithin has a strong impact on the chlorophyllase activity of Triticum. It was therefore investigated how chlorophyllase works in degumming with water in combination with different phospholipases / acyltransferases.

[0247] In this experiment, chlorophyllase from Triticum (see Example 1) and Chlamydomonas (see Example 2) were tested in combination with LysoMax Oil® (lipid acyl transferase from Danisco A / S), a phospholipase A1 (PLA1, Lecitase Ultra®) and a phospholipase C (PLC, Purifine®).

[0248] Degumming with water was carried out using the formula shown in Table 12. Table 12 1 2 3 4 5 6 7 8 Rapeseed oil g 10 10 10 10 10 10 10 10 water ml 0.310 0.300 0.290 0.300 0.085 0.075 0.065 0.075 Triticum CHL'ase, CoRe 20 ml 0.040 0.040 0.040 0.040 Chlamydomonas CHL'ase CoRe 31 ml 0.265 0.265 0.265 0.265 LysoMax Oil® 100 U / ml ml 10 10 Purifine® diluted 1:10 20 20 Lecitase Ultra® diluted 1:25 ml 10 10 Chlorophyllase U / g Oil 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 % Water 3.50 3.50 3.50 3.50 3.50 3.50 3.50 3.50

[0249] Crude rapeseed oil was heated to 55°C. Water and enzymes were added and the samples were homogenized with Petition 870260073365, dated 07 / 23 / 2026, page 74 / 198 68 / 92 high shear mixture for 20 seconds, followed by stirring with a magnetic stirrer. After 4 hours of incubation, the samples were heated to 97°C for 10 minutes and centrifuged at 1,780 fcr for 3 minutes.

[0250] The chlorophyll components of the oil phase were analyzed by HPLC with the results in Table 13 and Figure 27. Table 13 RFU Rel. 1.5 1.1 Tri CHL'ase Lee. Ultra 2.3 0.8 11.2 3.0 Chla CHL'ase Control 7.0 2.2 1.4 1.1 Chla CHL'ase LysoMax Oil® 3.3 1.2 8.8 3.5 Chla CHL'ase Purifine 7.8 2.4 1.3 0.8 Chla CHL'ase Lee. Ultra 3.8 1.1 7.6 2.9 - - 1.7 0.5 11.4 1.8

[0251] The results in Table 13 clearly indicate altered chlorophyllase activity in pheophytin in the presence of phospholipases or an acyltransferase. For comparison, in a control sample that does not comprise any enzyme, pheophytin A in crude rapeseed oil generates a fluorescence value of 11.4 RFU.

[0252] When chlorophyllases are combined with acyltransferase or PLA1, the amount of pheophytin is much higher than the control comprising chlorophyllase alone, but when chlorophyllases are combined with PLC, the pheophytin level is almost equal to the control oil treated only with chlorophyllase. Experiments indicate that chlorophyllases are inhibited when used in combination with enzymes that, during incubation, produce lysophospholipids from phospholipids. In contrast, chlorophyllases retain most of their original activity when used in combination with a PLC, which produces diglyceride from phospholipids. Petition 870260073365, dated 07 / 23 / 2026, page 75 / 198 69 / 92 Example 7 Chlorophylase in Total Degumming

[0253] Example 6 shows that chlorophyllase can be used during oil refining in the water degumming process. Depending on the type of oil and the refining process, different types of degumming processes can be used. Thus, as an alternative to a water degumming process, a plant oil can be refined without water degumming in a full degumming or neutralization process. In the following example, chlorophyllase was used in a degumming / neutralizing process. In this experiment, chlorophyllase is also tested in combination with acyltransferase with the formula shown in Table 14: Table 14 1 2 3 Raw rapeseed without 6 g 10 10 10 Water ml 0.253 0.243 0.293 TRI CHL CoRe 20 MRZ ml 40.0 40.0 0.0 Acyltransferase, LysoMax Oil®, 100U / ml ml 0.0 10.0 0.0 30% Phosphoric Acid μl 25.00 25.00 25.00 4M NaOH μl 47 47 47 Acyltransferase U / g of oil 0.0 0.1 0 Chl'ase Units / g of oil 0.050 0.050 0.000 % Water 3.500 3.500 3.500 Temperature °C 55 55 55 pH 6.5 6.4 6.3

[0254] Crude rapeseed oil was heated to 55°C. 30% phosphoric acid was added and the sample was homogenized with high shear mixing for 10 seconds followed by magnetic stirring at 55°C. After 10 minutes, water, NaOH and enzymes were added and incubated at 55°C with magnetic stirring. After 4 hours, the samples were heated to 97°C for 10 minutes and centrifuged at 1,780 fcr for 3 minutes. Petition 870260073365, dated 07 / 23 / 2026, page 76 / 198 70 / 92

[0255] The oil phase was analyzed by HPLC with the results shown in Table 15: Table 15 Phaeophorbid Pyropheophorbid Phaeophytin A Pyropheophytin Sample Enzyme RFU RFU RFU RFU 1 CI-L'ase 4.3 0.5 0.8 1.1 2 CHL'ase+LysoMaxOil® 3.8 0.4 5.4 1.6 3 Control 0.2 0.2 14.6 2.3

[0256] The results in Table 15 confirm the high chlorophyllase activity in pheophytin in a fully degumming oil process. In combination with acyltransferase, chlorophyllase activity is significantly reduced. Example 8 Oil Refining Procedure with Chlorophyllase, With and Without Bleaching

[0257] In this example, a Triticum chlorophyllase gene (called TRl_CHL, see Example 1) expressed in E. coli is used in the oil refining of crude rapeseed oil. This enzyme has activity on chlorophyll, pheophytin, and pyropheophytin in oil. In the first step, the oil is treated with TRI_CHL, whereby chlorophyll, pheophytin, and pyropheophytin are hydrolyzed to chlorophyllide, pheophorbide, and pyropheophorbide, respectively. After treatment with TRl_CHL, the oil is further refined without the use of bleaching clay, and the oil is compared with the same oil refined by traditional oil refining using bleaching clay. Oil Refining Procedure with Chlorophyllase and Without Bleaching

[0258] Degumming with water: 175 g of crude rapeseed oil are heated to 65°C during stirring and covered with nitrogen. 8.75 Units (0.05 Units / g) of TRI_CHL are added along with 6.125 g (3.5%) of water. The reaction mixture is homogenized with high-shear mixing for 20 seconds. A Petition 870260073365, dated 07 / 23 / 2026, page 77 / 198 The 71 / 92 sample is incubated at 65°C with agitation and covered with nitrogen. After a reaction time of 4 hours, the sample is centrifuged at 3,000 fcr for 5 minutes and the oil degummed with water is isolated.

[0259] Total degumming: 150 grams of water-degummed oil were dried by heating at 90°C for 20 minutes. The oil is cooled to 80°C and 0.33% phosphoric acid (30% aqueous solution) is added. The sample is homogenized by high shear mixing for 10 seconds and stirred for 10 minutes during cooling to 70°C followed by the addition of 1.28% 4N NaOH. The sample is stirred for 5 minutes covered with nitrogen. The sample is centrifuged at 3,000 fcr for 5 minutes.

[0260] Washing with NaOH: The oil is isolated and heated to 90°C. 4% 0.1N NaOH is added to the sample and stirred for 5 minutes. The oil is centrifuged at 3,000 rpm for 5 minutes.

[0261] Water washes: The isolated oil phase is heated to 50°C and washed with 4% water with agitation for 5 minutes and centrifuged at 300 rpm for 5 minutes. The oil phase is washed once more with 4% water. The isolated oil phase is dried by heating at 100°C under vacuum for 20 minutes.

[0262] Deodorization: The oil is deodorized at 240°C and 50 Pa (0.5 mbar) for 1 hour with steam draining. The oil is polished by filtration through a glass microfiber filter (Whatman GF / C). Reference Procedure for Oil Refining with Bleaching:

[0263] Degumming with water: 175 g crude rapeseed oil is heated to 65 °C during stirring and covered with nitrogen. 6.125 g (3.5%) of water is added. The reaction mixture is homogenized with high shear mixing for 20 seconds. The sample is incubated at 65 °C with stirring and covered with nitrogen. After a reaction time of 4 hours, the sample is centrifuged at 3,000 fcr for 5 minutes and the water-degummed oil is isolated. Petition 870260073365, dated 07 / 23 / 2026, page 78 / 198 72 / 92

[0264] Total degumming: 150 grams of water-degummed oil are dried by heating at 90°C for 20 minutes. The oil is cooled to 80°C and 0.33% phosphoric acid (30% aqueous solution) is added. The sample is homogenized by high-shear mixing for 10 seconds and stirred for 10 minutes during cooling to 70°C followed by the addition of 1.28% 4N NaOH. The sample is stirred for 5 minutes covered with nitrogen. The sample is centrifuged at 3,000 fcr for 5 minutes.

[0265] Bleaching: 1% bleaching clay (Tonsil Optimu FF210) is added to the oil and heated to 90°C under vacuum and stirring for 20 minutes. The oil is cooled to 80°C and filtered in a Büchner funnel using filter paper. The isolated oil phase is dried by heating to 100°C under vacuum for 20 minutes.

[0266] Deodorization: The oil is deodorized at 240°C and 50 Pa (0.5 mbar) for 1 hour with steam draining. The oil is polished by filtration through a glass microfiber filter (Whatman GF / 'C). Results

[0267] Crude rapeseed oil from AarhusKarlshamn was refined according to the above method using either (1) Chlorophyllase oil refining procedure without bleaching or (2) Reference oil refining procedure with bleaching.

[0268] The oil color was measured according to Lovi Bond using the Dr. Lange LICO 200 apparatus. The results are shown in Table 16: TABLE 16 Lovibond 51 / 4 Yellow Red Refined oil with bleaching 6.4 0.5 Refined oil without bleaching 16 0.2 Bunge specification for refined rapeseed oil (51 / 4 Lovibond) (http: / / www.bunge-austria.com / uploads / media / Spec_Rapeseed_Oleo_refined_A_Ol.pdf) Max 20 Max 1.5

[0269] HPLC / MS Analysis: Petition 870260073365, dated 07 / 23 / 2026, page 79 / 198 73 / 92

[0270] Oil samples from different processes in oil refining were analyzed by HPLC / MS and quantified with respect to component standards, with the results shown in Table 17: Table 17 Pheophorbidum Pyropheophorbidum Pheophytin b Pheophytin a Pyropheophytin ng / mg ng / mg ng / mg ng / mg ng / mg Oil refining with bleaching: After degumming with water 0.656 0.490 1.283 22.147 0.890 After total degumming 0.004 0.022 1.816 20.938 1.106 After bleaching 0.017 0.039 0.005 0.062 0.007 After deodorization 0.001 0.015 0.001 0.023 0.020 Oil refining with chlorophyllase: After degumming with water 2.547 0.845 0.027 0.855 0.254 After total degumming 0.034 0.033 0.037 0.963 0.254 After washing with NaOH 0.002 0.019 0.034 0.812 0.250 1. Washing with water 0.002 0.026 0.034 0.786 0.243 2. Washing with water 0.002 0.025 0.033 0.788 0.244 After deodorization 0.002 0.025 0.001 0.022 0.327

[0271] The results in Table 17 are illustrated graphically in Figures 29 and 30.

[0272] The results showed that chlorophyllase is active on pheophytin and pyropheophytin in the oil during water degumming. More than 95% of pheophytin and more than 70% of pyropheophytin are removed in the chlorophyllase-treated oil. These components are hydrolyzed into phytol and pheophorbide and pyropheophorbide, respectively. It is observed that after the water degumming process, these two components increase in the chlorophyllase-treated oil. However, a large proportion of these components is removed by washing with alkaline treatment in the process. Petition 870260073365, dated 07 / 23 / 2026, page 80 / 198 74 / 92 to completely degum. Subsequent washing with NaOH and water only helps to remove even more degradation products. Materials

[0273] In Examples 3 to 8, the following materials were generally used, except where otherwise specified: Oil: raw rapeseed oil extracted from AarhusKarlshamn

[0274] Enzymes: Triticum chlorophyllase expressed in E. coli and purified, CoRe20 identified (see Example 1); Chlorophylase of Chlamydomonas expressed in Bacillus, CoRe-31 identified (see Example 2); Lipid Acyltransferase, LysoMax Oil® from Danisco A / S, for example, comprising the amino acid sequence SEQ ID NO:23 Phospholipase C, Purifine® from Verenium Corporation Phospholipase A1, Lecitase Ultra® from Novozymes A / S

[0275] Emulsifiers: Sorbitan Monooleate, SMO from Danisco A / S Sorbitan trioleate, STO from Danisco A / S HPLC analysis

[0276] In Examples 3 to 7, chlorophyll derivatives were generally quantified by HPLC analysis according to the following method. The HPLC analysis was performed using a general method as described in Determination of chlorophylls and carotenoids by high-performance liquid chromatography during olive lactic fermentation. Journal of Chromatography, 585, 1991, 259 to 266.

[0277] The determination of pheophytin, pheophorbid, pyropheophytin and pyropheophorbid is performed by HPLC coupled to an array detector of Petition 870260073365, dated 07 / 23 / 2026, page 81 / 198 75 / 92 diode. The column used in the method is packed with C18 material and the chlorophylls were separated by gradient elution. Peaks are assigned using SigmaAldrich chlorophyll A and B standards, for example, based on the representative HPLC chromatogram from the Journal of Chromatography, 585, 1991, 259 to 266 shown in Figure 28. Example 9 Chlorophyllase Dosage in a Water Degumming Process

[0278] Triticum chlorophyllase was tested at different dosages in degumming with water of raw rapeseed oil according to the formula in Table 18.

[0279] The oil was heated to 65°C, water and enzyme were added. The sample was mixed with a high shear mixer for 20 seconds and incubated with magnetic stirring.

[0280] Samples were taken for analysis after 1 / 2, 1, and 2 hours and heated to 97°C for 10 minutes to inactivate the enzyme. The samples were then centrifuged at 10,000 fcr for 5 minutes and the chlorophyll components in the oil were analyzed by HPLC / MS. Table 18 1 2 3 4 5 6 Raw rapeseed without water 8 g 10 10 10 10 10 10 ml 0.350 0.348 0.345 0.338 0.315 0.232 Triticum Chlorophyllase 14 U / ml ml 0.0024 0.0047 0.0118 0.0355 0.1183 Oil Units / g 0.000 0.005 0.010 0.025 0.075 0.250 % water 3.500 3.500 3.500 3.500 3.500 3.500 Temperature °C 65 65 65 65 65 65 The HPLC results are shown in Table 19: Table 19 ng / mg = pg / g ID Time Phaeophorbid Pyropheophorbid Phaeophytin b Phaeophytin a Pyropheophytin Chlorophyll b Chlorophyll a 2516-186-1 % 0.27 0.23 0.48 9.41 0.60 0.63 1.12 Petition 870260073365, dated 07 / 23 / 2026, page 82 / 198 76 / 92 ng / mg = pg / g ID Hour Pheophorbide Pyropheophorbide Pheophytin b Pheophytin a Pyropheophytin Chlorophyll b Chlorophyll a 2516-186-2 % 0.79 0.27 0.34 6.96 0.57 0.15 0.30 2516-186-3 % . 0.81 0.26 0.30 6.25 0.57 0.12 0.25 2516-186-4 % 0.96 0.29 0.22 5.02 0.53 0.11 0.22 2516-186-5 % 1.09 0.31 0.17 4.00 0.49 0.08 0.18 2516-186-6 % 1.25 0.37 0.08 2.12 0.39 0.04 0.10 2516-186-1 1 0.41 0.26 0.48 9.47 0.63 0.34 0.66 2516-186-2 1 0.98 0.24 0.24 5.32 0.57 0.08 0.17 2516-186-3 1 0.97 0.27 0.21 4.56 0.55 0.07 0.16 2516-186-4 1 1.11 0.31 0.13 3.20 0.51 0.05 0.13 2516-186-5 1 1.28 0.41 0.09 2.39 0.44 0.04 0.09 2516-186-6 1 1.39 0.45 0.04 1.22 0.31 0.01 0.03 2516-186-1 2 0.53 0.25 0.45 9.06 0.66 0.14 0.28 2516-186-2 2 1.25 0.31 0.14 3.36 0.53 0.03 0.08 2516-186-3 2 1.28 0.32 0.11 2.68 0.52 0.02 0.07 2516-186-4 2 1.29 0.33 0.05 1.74 0.43 0.02 0.06 2516-186-5 2 1.41 0.39 0.04 1.29 0.35 0.01 0.03 2516-186-6 2 1.44 0.54 0.02 0.66 0.17 0.01 0.02

[0281] Each of the four chlorophyll derivatives aebe pheophytin aeb exists as a pair of epimers determined by H and COOCH3 stereochemistry about carbon number 132 (numbering according to the IUPAC system). These are denoted a / be a' / b' with the initiator forms (') having S-stereochemistry and the non-initiator forms having Restereochemistry.

[0282] From the results in Table 19, it is clear that the main component in this oil is pheophytin. Pheophytin is normally the main green component in oil, due to the fact that chlorophyll readily loses its magnesium and becomes pheophytin. Because pheophytin is the main component, it is chosen to focus on this component for enzymatic degradation analysis.

[0283] The effect of chlorophyllase as a function of dosage of Petition 870260073365, dated 07 / 23 / 2026, page 83 / 198 77 / 92 enzyme and reaction time is illustrated in Figure 33. Initially (1 / 2 hour) there is a strong reduction in the amount of pheophytin even at a low enzyme dosage and then the enzyme activity decreases over time, but the amount of pheophytin still decreases after a feeding time of 2 hours.

[0284] The results in Table 19 confirm the chlorophyllase activity of Triticum on the degradation of chlorophyll, pheophytin and pyropheophytin. The results of the control sample without enzyme addition, however, reveal that chlorophyll aeb are thermally degraded, most likely due to the fact that chlorophyll loses magnesium and is converted into pheophytin.

[0285] It is also observed that the amount of pyropheophytin increases as a function of time in the control sample, most likely due to the fact that some of the pheophytin is converted into pyropheophytin. The formation of pyropheophytin is not preferential due to the fact that the enzyme activity in this component is much lower than in pheophytin. The product of the hydrolysis reaction of pyropheophytin is pyropheophorbide, which is more hydrophobic than pheophorbide and thus more difficult to remove by washing the oil. Example 10 Effect of pH and Water Concentration on Chlorophylase Activity

[0286] In this study, Triticum chlorophyllase (TRl_CHL) was investigated in a complete degumming process with different water dosages and different pH adjustments according to the formula in Table 20. Table 20 2516-190- 1 2 3 4 5 6 7 8 9 10 11 12 13 Raw rapeseed, 6 g 10 10 10 10 10 10 10 10 10 10 10 10 10 Petition 870260073365, dated 07 / 23 / 2026, p. 84 / 198 78 / 92 2516-190- 1 2 3 4 5 6 7 8 9 10 11 12 13 water ml 0.135 0.111 0.087 0.039 0.285 0.261 0.237 0.189 0.435 0.411 0.387 0.339 0.261 TRI CHL CoRe 52 21.14 U / ml ml 23.7 23.7 23.7 23.7 23.7 23.7 23.7 23.7 23.7 23.7 23.7 23.7 0.0 2516-190- 1 2 3 4 5 6 7 8 9 10 11 12 13 NaOH 1M ml 50.0 75.0 100.0 150.0 50.0 75.0 100.0 150.0 50.0 75.0 100.0 150.0 100.0 Citric acid, 50% solution ml 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 Units / g of oil 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.000 % of Water 2,000 2,000 2,000 3,500 3,500 3,500 5,000 5,000 5,000 5,000 5,000 5,000 3,500 Temperature °C 55 55 55 55 55 55 55 55 55 55 55 55 55 pH 3.9 5.2 6.1 6.9 4.7 5.3 6.1 7.0 5.0 5.6 5.9 6.9 6.1 pmol NaOH 0.05 0.075 0.1 0.15 0.05 0.075 0.1 0.15 0.05 0.075 0.1 0.15 0.1 pmol citric acid 0.033 0.033 0.033 0.033 0.033 0.033 0.033 0.033 0.033 0.033 0.033 0.033 0.033 Ratio mol NaOH / citric acid 1.500 2.250 3.000 4.500 1.500 2.250 3.000 4.500 1.500 2,250 3,000 4,500 3,000.

[0287] The oil was heated to 55°C and citric acid was added. The sample was mixed with the high-shear mixture for 20 seconds followed by magnetic stirring for 10 minutes. NaOH, water, and enzyme were added and mixed for 20 seconds with the high-shear mixture. The samples were incubated with magnetic stirring for 4 hours. Samples were taken for analysis after 1 / 2, 1, and 2 hours, and heated to 97°C for 10 minutes to inactivate the enzyme. The samples were then centrifuged at 10,000 fcr for 5 minutes, and the oil phase was analyzed by HPLC / MS.

[0288] The effect of pH and % water on chlorophyllase activity is illustrated in Figure 34. At 2% water in the reaction mixture, chlorophyllase activity increases with high pH up to almost pH 7. The same trend is also seen for 3.5% water, but in the experiment with 5% water, enzyme activity increased at pH 6, but between 6 and 7 the activity decreases again. Petition 870260073365, dated 07 / 23 / 2026, page 85 / 198 79 / 92

[0289] It is known that both chlorophyll and pheophytin exist in two epimeric forms, a and a' (R isomer and S isomer), and the enzyme chlorophyllase is only active in the a isomer form. It is known that an equilibrium exists between the two forms and the rearrangement is pH dependent (Figure 35).

[0290] It is therefore expected that enzyme activity will be pH-dependent due to the fact that higher pH will favor the formation of the α isomer. Based on HPLC analysis of pheophytin epimers αea', it is possible to calculate the ratio of the α epimer at different pH values ​​as shown in Figure 36. The results in Figure 36 confirm that an increase in pH will favor the formation of the α epimer, which can then explain the elevated enzyme activity at higher pH. Part of this increase in activity can also be explained by other factors such as enzyme activity at different pH values. Example 11 Effect of Reaction Temperature on Chlorophylase Activity

[0291] The experiments mentioned above were conducted at 55°C because this is the temperature typically used in water degumming with enzymes (e.g., LysoMax Oil®). Triticum chlorophyllase (TRI_CHL), however, is known to be more heat-stable, and in the following experiment the enzyme activity in the oil was investigated at 65, 70, and 75°C according to the formula in Table 21. Table 21 2516-194- 1 2 3 4 5 6 7 8 9 10 11 12 Raw rapeseed without 8 10 10 10 10 10 10 10 10 10 10 10 10 water ml 0.350 0.334 0.318 0.302 0.350 0.334 0.318 0.302 0.350 0.334 0.318 0.302 TRI_CHL CoRe 70-10.31 Uml ml 0.0161 0.0323 0.0484 0.0000 0.0161 0.0323 0.0484 0.0000 0.0161 0.0323 0.0184 Units / g of oil 0.000 0.050 0.100 0.150 0.000 0.050 0.100 0.150 0.000 0.050 0.100 0.150 % of water 3.50 3.50 3.50 3.50 3.50 3.50 3.50 3.50 3.50 3.50 3.50 3.50 Temperature °C 65 65 65 65 70 70 70 70 75 75 75 75 Petition 870260073365, dated 07 / 23 / 2026, page 86 / 198 80 / 92

[0292] The oil was heated to the fixation point of 65, 70, or 75 °C. Water and enzyme were added, and the sample was mixed with the high-shear mixture for 20 seconds and incubated with stirring at a defined temperature. Samples were taken for analysis after 1 / 2, 1, and 2 hours and heated to 97 °C for 10 minutes to inactivate the enzyme. The samples were then centrifuged at 10,000 fcr for 5 minutes, and the oil phase was analyzed by HPLC / MS. The results were statistically evaluated using the ANOVA Statgraphic software.

[0293] The effect of temperature on pheophytin levels is shown in Figure 37. The results confirm that activity decreases at 75°C, but there is no clear difference in enzyme activity at 65 and 70°C.

[0294] The results indicate that the enzymatic degradation of green color is not limited to the degradation of pheophytin, but pyropheophytin is also an important component. One of the limitations is that Triticum chlorophyllase has much lower activity on pyropheophytin compared to its activity on pheophytin, and therefore more enzyme and / or reaction time is required to hydrolyze this component. Another aspect is that pheophytin can be converted into pyropheophytin, and it could be expected that temperature would have an impact on this.

[0295] The effect of temperature on pyropheophytin levels is shown in Figure 38. The results clearly indicate that the amount of pyropheophytin is higher in samples incubated at higher temperatures. This could obviously be explained by the lower enzyme activity, but it was shown that the enzyme activity on pheophytin at 70°C was at least leveled with the activity at 65°C. The results, therefore, indicate that more pyropheophytin is produced at higher temperatures.

[0296] Taking into account that part of the chlorophyll is converted into pheophytin and pheophytin is converted into pyropheophytin, the effect of temperature Petition 870260073365, dated 07 / 23 / 2026, page 87 / 198 The activity of the enzyme on these three components, as measured by Figure 39, was also investigated (Figure 39). It is calculated that the enzyme activity on these three components is at the same level at 65 and 70°C, but at 75°C the enzyme activity is lower. Example 12 Mixing Conditions During Chlorophyllase Activity in Oil / Water

[0297] The enzymatic reaction of Triticum chlorophyllase (TRI_CHL) into chlorophyll in oil is conducted in a two-phase oil / water reaction mixture. It could therefore be speculated that the reaction might depend on the water distribution and particle size of the water droplets. In order to investigate this in more detail, experiments were set up with and without the high-shear mixture. In addition, experiments were set up in which enzyme and water were mixed before addition to the oil (Table 22). In all experiments, 3.5% water was used. Table 22 1 2 3 4 5 6 7 8 Raw rapeseed without 8 10 10 10 10 10 10 10 10 water ml 0.350 0.334 0.334 0.484 0.484 0.334 0.334 0.334 TRI_CHL CoRe 70-10.( U / ml) ml 0.0161 0.0161 0.0161 0.0161 0.0161 0.0161 0.0161 Units / g of oil 0.000 0.050 0.050 0.050 0.050 0.050 0.050 0.050 % of water 3.500 3.500 3.500 3,500 3,500 3,500 3,500 3,500 Temperature °C 65 65 65 65 65 65 65 65 Ultra Turrax 20 seconds + - + - + + - + Ultra Turrax Interval 5 seconds every 10 minutes + Enzyme + water mixed before addition + +

[0298] The oil was heated to 65°C with magnetic stirring. Enzyme and water were added. Samples were treated with high shear mixing according to Table 22 and incubated with magnetic stirring. 1 ml samples were taken after reaction times of 1 / 2, 1 Petition 870260073365, dated 07 / 23 / 2026, page 88 / 198 82 / 92 and 2 hours. The samples were then centrifuged at 10,000 fcr for 5 minutes and the oil phase was analyzed by HPLC / MS.

[0299] The results for pheophytin content are graphically illustrated in Figure 40, which shows that there is not much difference in the pheophytin level in the samples treated with or without high shear mixing (Ultra Turrax). This indicates that less vigorous mixing with a magnetic stirrer is sufficient to obtain a good enzyme reaction and this cannot be improved by initial high shear mixing which produces much finer water droplets in the reaction mixture. Example 13 pH Effect on Water Degumming

[0300] The experiments shown above (Example 10) indicated that chlorophyllase activity and also the rearrangement of pheophytin a' into pheophytin a was pH dependent. In the following experiment, the degumming process with water was conducted both without pH adjustment and with pH adjustment using NaOH or citrate buffer. In one experiment, an acyltransferase was also tested in combination with chlorophyllase. The oil was heated to 65°C and water, NaOH / buffer and enzyme were added.

[0301] Samples were mixed with Ultra Turrax for 20 seconds and incubated at 65°C with magnetic stirring. Samples were taken after reaction times of 1 / 2, 1, 2, and 4 hours. Samples were heated to 95°C for 10 minutes to inactivate the enzyme, and centrifuged at 10,000 fcr for 5 minutes, and the oil phase analyzed by HPLC / MS. Table 23 1 2 3 4 5 6 7 8 9 Raw rapeseed without water 8 10 10 10 10 10 10 10 10 ml 0.200 0.186 0.180 0.166 0.170 0.156 0.050 0.036 0.146 1N NaOH 0.020 0.020 0.030 0.030 0.030 Petition 870260073365, dated 07 / 23 / 2026, page 89 / 198 83 / 92 1 2 3 4 5 6 7 8 9 Citrate a mM pH 6 0.150 0.150 LysoMax Oil® 0.010 TRI_CHL CoRe 70_11 (69.77 U / ml) ml 0 0.0143 0.0000 0.0140 0.0000 0.0143 0.0000 0.0143 0.014 Units / g of oil 0.000 0.100 0.000 0.100 0.000 0.100 0.000 0.100 0.100 % water 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Temperature °C 65 65 65 65 65 65 65 65 65 pH 5.21 5.17 7.21 7.12 7.50 7.48 5.50 5.40 7.43

[0302] In Figure 41, the amount of pheophytin (a+a') is illustrated as a function of time and different pH conditions. It was observed that increasing the pH above 7 with the addition of NaOH will increase the enzyme activity on pheophytin. This is probably explained by the fact that the rearrangement of pheophytin a' to pheophytin a is pH dependent.

[0303] In Figure 42, the ratio of epimer a is illustrated and it is very clear that at pH 5.12 and 5.4 the amount of epimer a is low due to the fact that chlorophyllase is only active on this epimer and the redistribution is slow due to the higher pH. At pH 7.12 and 7.48, the amount of epimer a is almost maintained at the equilibrium concentration (approximately 70%) throughout the process. Only after a reaction time of 4 hours does the relative amount of epimer a decrease, probably due to the fact that the total amount of pheophytin is then very low.

[0304] Although it is very important that the enzyme is active on pheophytin, it is also important for the green color that the process can remove the other components of including pyropheophytin. Figure 43 illustrates the amount of pyropheophytin in the sample treated with Triticum chlorophyllase at different pH. It can be concluded that the lower pH has a positive effect on the removal of pyropheophytin. This can be explained by the lower enzyme activity on pyropheophytin at higher pH or by the formation of pyropheophytin, Petition 870260073365, dated 07 / 23 / 2026, pp. 90 / 198 84 / 92 which is catalyzed by a higher pH.

[0305] In Figure 44, the amount of pyropheophytin is subtracted from the amount of pyropheophytin in the control sample, in which no chlorophyllase is added. This graph indicates that a change in pyropheophytin caused by the enzyme is almost the same at different pHs. It is concluded, then, that the higher pH promotes the formation of pyropheophytin from pheophytin, which explains the results in Figure 44. Example 14 Effect of pH on Chlorophyllase Activity in Oil-Water Degumming.

[0306] The results reported above indicate the effect of pH on enzyme activity and the rearrangement of pheophytin a' into pheophytin a. The higher pH in the process also appears to have an impact on the conversion of pheophytin to pyropheophytin. In this study, pH was further investigated by narrowing the pH range for the degumming tests with Triticum chlorophyllase water (TRI_CHL). The experiments were conducted according to Table 24. Table 24 1 2 3 4 5 6 7 8 Raw rapeseed without water 8 10 10 10 10 10 10 10 10 ml 0.200 0.182 0.172 0.157 0.142 0.117 0.102 0.082 1N NaOH ml 0.010 0.025 0.040 0.065 0.080 0.100 TRI_CHL CoRe 70_11.(54.19U / ml) ml 0 0.0185 0.0185 0.0185 0.0185 0.0185 0.0185 0.0185 Units / g of oil 0.000 0.100 0.100 0.100 0.100 0.100 0.100 0.100 % water 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 Temperature °C 65 65 65 65 65 65 65 65 pH 5.03 4.78 5.60 5.99 6.32 6.75 7.06 7.30

[0307] The oil was heated to 65 °C and water, NaOH and enzyme were added. The samples were mixed with Ultra Turrax for 20 seconds. Petition 870260073365, dated 07 / 23 / 2026, page 91 / 198 85 / 92 and incubated at 65°C with magnetic stirring. Samples were taken after reaction times of 1 / 2, 1, 2, and 4 hours. Samples were heated to 95°C for 10 minutes to inactivate the enzyme, and centrifuged at 10,000 fcr for 5 minutes, and the oil phase was analyzed by HPLC / MS.

[0308] The effect of adding NaOH and adjusting the pH had an impact on the enzyme's ability to degrade pheophytin (see Figure 45). At pH 4.5 to pH 6, the enzyme activity appears to be at the same level. There is even a tendency for decreased enzyme activity going from pH 4.5 to pH 6 after a reaction time of ½ hour, but these levels fall with prolonged reaction time. Above pH 6, there is a clear reduction in the pheophytin level indicating high enzyme activity, and the ideal pH for the reaction is between pH 6.3 and 6.8.

[0309] One possible explanation for the effect of pH on chlorophyllase activity is the fact that the enzyme is only active on the α epimer of pheophytin (R isomer) and not on the α' epimer of pheophytin (the S isomer). The graphs in Figure 46 illustrate the effect of pH on the relative amount of α epimer of pheophytin. It is very clear that increasing the pH from 4.5 to 7 will result in a higher amount of the α epimer, going from about 20% to about 70% of an α epimer, which is the equilibrium concentration. The change in the ratio of the α epimer is explained by the fact that the lower pH (higher H concentration) prevents the rearrangement from moving towards equilibrium.

[0310] Triticum chlorophyllase has much lower activity on pyropheophytin than on pheophytin. In the reaction of chlorophyllase with oil, it is therefore also important that the process be optimized to produce the least possible amount of pyropheophytin. Figure 47 illustrates the amount of pyropheophytin as a function of pH.

[0311] The results clearly show a decrease in Petition 870260073365, dated 07 / 23 / 2026, page 92 / 198 86 / 92 pyropheophytin as a function of reaction time, but it is also observed that the sample with the higher pH contains more pyropheophytin. This can be explained by the conversion of pheophytin to pyropheophytin, which is promoted by the higher pH.

[0312] In selecting the ideal pH condition for the enzymatic degradation of chlorophyll components, it is important not only to observe the enzyme kinetics, but also to observe the different epimers of pheophytin and the conversion of pheophytin to pyropheophytin. In Figure 48, the effect of chlorophyllase on the amount of pheophytin + pyropheophytin is illustrated as a function of pH.

[0313] The results in Figure 48 confirm that pH 6.3 to 6.8 is the best range for the process. However, it is also seen that after a reaction time of 4 hours there is no strong effect of pH on the degradation of pheophytin plus pyropheophytin.

[0314] During sample preparation for HPLC / MS analysis, the samples were centrifuged and the oil phase was analyzed. The reaction products for pheophytin hydrolysis will then be distributed between the oil and water phases. The residual amount of pheophorbide in the oil phase as a function of pH is illustrated in Figure 49. The graphs in Figure 49 confirm that the amount of pheophorbide drops drastically when the pH increases. This is explained by the fact that the elevated pH converts pheophorbide into its ionized salt form, which is much more soluble in water. Example 15 Effect of pH on Chlorophyllase Activity in the Complete Degumming Process

[0315] In the examples above, it was observed that it could be beneficial to adjust the pH in the degumming process with water when using Petition 870260073365, dated 07 / 23 / 2026, page 93 / 198 87 / 92 Triticum chlorophyllase (TRI_CHL). In the complete degumming process, acids and alkalis are always added during the process. In the following experiments, the effect of pH on the activity of Triticum chlorophyllase in the complete degumming process was investigated first by treating the oil with citric acid by adding different amounts of NaOH according to Table 25. Table 25 2516-208- 1 2 3 4 5 6 7 8 Raw rapeseed without water 8 10 10 10 10 10 10 10 10 ml 0.036 0.097 0.072 0.052 0.037 0.018 0.000 0.000 Citric acid, 50% solution ml 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.020 1N NaOH ml 0.160 0.078 0.104 0.125 0.140 0.160 0.180 0.198 TRI_CHL CoRe 70_11 (54.19 (U / ml) ml 0 0.0185 0.0185 0.0185 0.0185 0.0185 0.0185 0.0185 Units / g of oil 0.000 0.100 0.100 0.100 0.100 0.100 0.100 0.100 % of water 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Temperature °C 65 65 65 65 65 65 65 65 pH 6.34 3.99 4.30 5.20 5.87 6.16 6.54 6.59

[0316] The oil was heated to 65°C and citric acid was added. The samples were mixed with Ultra Turrax for 20 seconds and incubated at 65°C with magnetic stirring for 10 minutes. NaOH, water, and enzyme were added, and the samples were mixed with Ultra Turrax for 20 seconds, followed by incubation at 65°C with magnetic stirring. Samples were taken after reaction times of 1 Λ, 1, and 2 hours. The samples were heated to 95°C for 10 minutes to inactivate the enzyme, and centrifuged at 10,000 fcr for 5 minutes, and the oil phase analyzed by HPLC / MS.

[0317] In this example of total degumming, the same tendency is Petition 870260073365, dated 07 / 23 / 2026, page 94 / 198 88 / 92 observed that in the water degumming process. That is, Triticum chlorophyllase is more active in pheophytin when the pH is raised from 4 to 6.6, which again is linked to the amount of the two epimer forms of pheophytin. The experiments mentioned above with water degumming were conducted with the same enzyme and oil dosage as in the total degumming process, and then the results of pheophytin degradation after a reaction time of two hours as a function of pH in the two processes were compared as shown in Figure 50. The graphs in Figure 50 indicate that the effect of pH adjustment is even stronger in total degumming than in water degumming. This could be explained by the addition of citric acid, which increases the ionic resistance in the water phase and also has an impact on the hydration of phospholipids. Example 16 Effect of Water Content and pH on Chlorophylase Activity in Oil

[0318] Previous studies have shown that Triticum chlorophyllase (TRl_CHL) required at least 1.5% water in oil for good pheophytin activity. Under certain conditions, however, it might be preferable to use a lower water content. In the following experiments, the degumming process with water was conducted with 1 and 2% water and with the addition of different NaOH as shown in Table 26. Table 26 2516-208- 1 2 3 4 5 6 7 8 9 10 11 12 Raw rapeseed without water 8 10 10 10 10 10 10 10 10 10 10 10 10 ml 0.082 0.057 0.042 0.017 0.182 0.157 0.14 2 0.11 7 0.100 0.035 0.200 0.135 NaOH 1N ml 0.02 5 0.04 0 0.065 0.025 0.04 0 0.065 0.065 0.065 TRI_CHL CoRe 70_11 (54.19 U / ml) ml 0.018 5 0.018 5 0.018 5 0.018 5 0.018 5 0.018 5 0.018 5 0.018 5 0.000 0 0.000 0 0.000 0 0.000 0 Petition 870260073365, dated 07 / 23 / 2026, pp. 95 / 198 89 / 92 Units / g of oil 0.100 0.100 0.100 0.100 0.100 0.100 0.100 0.000 0 0.000 0 0.000 0 0.000 0 % of water 1.000 1.000 1.000 1.000 2.000 2.000 2.000 2.000 1.000 1.000 2.000 2.000 Temperature °C 65 65 65 65 65 65 65 65 65 65 65 65 pH 4.64 5.47 5.69 6.14 5.12 5.85 6.23 6.78 4.70 6.35 4.63 6.79

[0319] The process was conducted according to the standard conditions mentioned in Example 14 and samples taken after 2 and 4 hours were analyzed by HPLC / MS.

[0320] The effect of pH and water content on the amount of pheophytin in chlorophyllase-treated oil is illustrated in Figure 51 and Figure 52. The results clearly show that Triticum chlorophyllase is active in pheophytin in a process with 1% water, and the activity is better at 1% water compared to 2% water. The higher pheophytin activity at 1% water cannot be explained by higher conversion to pyropheophytin (Figure 52) or by the rearrangement of the α' epimer to α epimer (Figure 53). It would then be speculated that the enhanced activity at 1% water is explained by different physical properties (mesomorphic properties) of the polar lipids at 1% water compared to 2% water, which in turn changes the enzyme's inactivity or substrate accessibility. Example 17 Temperature Optimization for Oil Chlorophyllase Treatment

[0321] Studies have shown that Triticum chlorophyllase is active in oil at temperatures above 70°C. At 70°C, the enzyme has its maximum activity, but at this temperature the conversion of pheophytin to pyropheophytin increases significantly. Previous studies concluded that a reaction temperature of 65°C was better than 70°C. In this study, the reaction temperature was further investigated by performing the enzyme reaction and degumming process with water at 60 and 65°C with variation in enzyme dosage and pH adjustment according to the experiments shown in Petition 870260073365, dated 07 / 23 / 2026, pp. 96 / 198 90 / 92 Tables 27 and 28. Table 27 2510-018- 1 2 3 4 5 6 7 8 Raw rapeseed without water 8 10 10 10 10 10 10 10 10 ml 0.200 0.190 0.170 0.100 0.152 0.142 0.122 0.052 NaOH 1N ml 0.050 0.050 0.050 0.050 TRI_CHL CoRe 70_11 ml 0.0100 0.0300 0.1000 0.0100 0.0300 0.1000 Units / g of oil ml 0.000 0.005 0.015 0.050 0.000 0.005 0.015 0.050% water 2.000 2.000 2.000 2.000 2.000 2.000 2.000 Temperature °C 65 65 65 65 65 65 65 65 Table 28 2510-018- 9 10 11 12 13 14 15 16 Raw rapeseed without water 8 10 10 10 10 10 10 10 10 ml 0.200 0.190 0.170 0.100 0.152 0.142 0.122 0.052 NaOH 1N ml 0.050 0.050 0.050 0.050 TRI_CHL CoRe 70_11 ml 0.0100 0.0300 0.1000 0.0100 0.0300 0.1000 Units / g of oil ml 0.000 0.005 0.015 0.050 0.000 0.005 0.015 0.050 % water 2.000 2.000 2.000 2.000 2.000 2.000 2.000 Temperature °C 60 60 60 60 60 60 60 60

[0322] The oil was heated to 65°C / 60°C. NaOH, water, and enzyme were added, and the samples were mixed with Ultra Turrax for 20 seconds, followed by incubation with magnetic stirring. Samples were taken after a reaction time of 2 and 4 hours. The samples were heated to 95°C for 10 minutes to inactivate the enzyme, and centrifuged at 10,000 fcr for 5 minutes, and the oil phase analyzed by HPLC / MS. The results are graphically illustrated in Figures 54 to 56.

[0323] The results in Figure 54 confirm that chlorophyllase activity is correlated with enzyme dosage, but even a dosage of 0.005 U / g of enzyme has a significant effect on pheophytin. It is also observed that the activity at 60°C is at least leveled with the activity at 65°C. pH adjustment in the process also appears to have an effect. Petition 870260073365, dated 07 / 23 / 2026, pp. 97 / 198 91 / 92 positive on enzyme activity, which is most likely explained by the change in epimer rearrangement when pH is elevated (Figure 56).

[0324] There is also some indication that the level of pyropheophytin is lower at 60°C (Figure 55).

[0325] The results taken together indicated that 60°C is a preferred reaction temperature for Triticum chlorophyllase when used in a water degumming process. Conclusion

[0326] Triticum chlorophyllase has been shown to be active on the three main chlorophyll components, chlorophyll, pheophytin and pyropheophytin in an oil system. The enzyme activity was dependent on numerous process parameters including temperature, pH, mixing, % water and reaction time.

[0327] Experiments showed that pH 6.3 to 6.5 was the best range for Triticum chlorophyllase activity due to the fact that the enzyme is only active on the α' epimer of pheophytin. At pH 6.3 to 6.5, rearrangement of α' of pheophytin into α epimer can occur in the process. At higher pH, a higher rearrangement of α' pheophytin into α is observed, but it is also observed that at pH higher than 6.5 more pyropheophytin is produced from pheophytin. This is not preferential due to the fact that Triticum chlorophyllase is less active on pyropheophytin.

[0328] It was revealed that the conversion of pheophytin to pyropheophytin is also temperature dependent. At 70°C and above, significantly more pyropheophytin is produced from pheophytin, and this is not preferential. Experiments showed that the ideal temperature for Triticum chlorophyllase was 60°C, which is the best common denominator between enzyme kinetics and the conversion of pheophytin to pyropheophytin.

[0329] In a degumming process with normal water, typically 2% water is used and experiments have shown that the Petition 870260073365, dated 07 / 23 / 2026, pp. 98 / 198 92 / 92 Triticum chlorophyllase was highly active in 2% water. It was revealed that the enzyme was even more active in 1% water. This lower water concentration could, in some cases, be advantageous if the viscosity of the resulting gum phase from this process is not too high for proper handling and pumping.

[0330] All publications mentioned in the descriptive report above are incorporated into the present invention by reference. Various modifications and variations of the methods and systems described in the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. In fact, various modifications of the described modes for carrying out the invention that are obvious to those skilled in biochemistry and biotechnology or related fields are intended to fall within the following claims. Petition 870260073365, dated 07 / 23 / 2026, pp. 99 / 198

Claims

1 / 2 Claims 1. PROCESS FOR REFINING A PLANT OIL, characterized by comprising a step of bringing the oil into contact with the enzyme Triticum aestivum chlorophyllase of SEQ ID No. 2, which is capable of hydrolyzing chlorophyll or a chlorophyll derivative, wherein the enzyme is brought into contact with the oil: (i) in the presence of at least 1% by weight of phospholipid; (ii) in the presence of less than 0.2% by weight of lysophospholipid; (iii) at a temperature of 58°C to 62°C; (iv) at a pH of 6.3 to 6.5; (v) in the presence of 1% to 2% by weight of water; and (vi) before or during a degumming step of the oil.

2. PROCESS, according to claim 1, characterized by comprising (a) bringing the enzyme into contact with the oil prior to (b) degumming the oil using a phospholipase or an acyltransferase.

3. PROCESS, according to claim 1, characterized by comprising bringing the oil into contact with the enzyme and a phospholipase C in a single step.

4. PROCESS, according to any one of claims 1 to 3, characterized in that the degumming step comprises degumming with water.

5. PROCESS, according to any one of claims 1 to 4, characterized in that the degumming step comprises adding an acid to the oil, followed by neutralization with an alkali.

6. PROCESS, according to any of claims 1 to 5, characterized by not comprising a step of Petition 870260073365, dated 07 / 23 / 2026, page 100 / 198 2 / 2 treatment with clay.

7. PROCESS, according to any one of claims 1 to 6, characterized by further comprising carrying out a deodorization step to produce a deodorized oil and a distillate. Petition 870260073365, dated 23 / 07 / 2026, pp. 101 / 198

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