Processing methods of vegetable oil

Abstract

The present invention provides a method for processing vegetable oil, comprising the step of contacting the oil with an enzyme, wherein the enzyme is capable of hydrolyzing chlorophyll or chlorophyll derivatives of a' or b' stereoisomers.

Description

[0001] This application is a divisional application of the invention patent application filed on February 16, 2012, with application number 201280009244.5 and entitled "Processing Method". Technical Field

[0002] This invention relates to the industrial processing of plant-based food and feed products, particularly vegetable oils. This invention can be used to reduce or eliminate contamination caused by chlorophyll and chlorophyll derivatives. Background Technology

[0003] Chlorophyll is a green pigment widely distributed throughout the plant kingdom. It is essential for photosynthesis and is one of the most abundant organometallic compounds found on Earth. Consequently, many plant-derived products (including food and feed) contain significant amounts of chlorophyll.

[0004] For example, vegetable oils derived from oilseeds (such as soybeans, palm or rapeseed (canola), cottonseed) and peanut oils typically contain some chlorophyll. However, high levels of chlorophyll pigment are generally not expected in vegetable oils. This is because chlorophyll gives the oil its unsightly green color and can cause oxidation during storage, leading to spoilage.

[0005] Various methods have been employed to remove chlorophyll from vegetable oils. Chlorophyll can be removed at multiple stages of the oil production process, including seed crushing, oil extraction, degumming, alkali treatment, and bleaching. However, the bleaching step is generally the most effective for reducing chlorophyll residues to acceptable levels. During bleaching, the oil is heated and passed through an adsorbent to remove chlorophyll and other colored compounds that affect the appearance and / or stability of the finished oil. The adsorbent used in the bleaching step is typically clay.

[0006] In the edible oil processing industry, the above steps typically reduce 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 the entrainment effect of the bleaching clay. The use of clay can remove many beneficial compounds from the oil (such as carotenoids and tocopherols). Furthermore, the use of clay is expensive, especially because the disposal of used clay (i.e., waste) is difficult, hazardous (prone to spontaneous combustion), and therefore costly. Therefore, attempts have been made to remove chlorophyll from the oil using other methods, such as the use of chlorophyllase.

[0007] In plants, chlorophyllase (or chlase) is believed to participate in chlorophyll degradation and catalyze the hydrolysis of ester bonds in chlorophyll to produce phloroglucinol and phytosterol. WO 2006009676 describes an industrial method for reducing chlorophyll contamination in compositions, such as treating vegetable oils with chlorophyllase. The water-soluble phloroglucinol produced in this method is also green, but can be removed by water extraction or silica treatment.

[0008] Chlorophyll is typically partially degraded in seeds used for oil production and during oil extraction from seeds. A common modification involves the loss of magnesium ions from the porphyrin (dihydroporphyrin) ring to form a derivative called pheophytin (see [link to original text]). Figure 1 The loss of highly polar magnesium ions from the porphyrin ring results in pheophytin being significantly different in physicochemical properties from chlorophyll. Typically, during processing, pheophytin is more abundant than chlorophyll in oil. Phthaphyllin is pale green and can be removed from oil by methods similar to those used for chlorophyll, such as the esterase reaction catalyzed by an enzyme with pheophytinase activity as described in WO 2006009676. Under certain conditions, some chlorophyllases can hydrolyze both pheophytin and chlorophyll, thus being suitable for removing these two contaminants. The products of pheophytin hydrolysis are red / brown pheophytic acid and phytol. Phthatic acid can also be generated due to the loss of magnesium ions from phytophytic chlorophyll (i.e., after chlorophyll hydrolysis) (see...). Figure 1 WO 2006009676 teaches the removal of pheophytic acid by a method similar to that of deplanting chlorophyll (e.g., by water extraction or silica adsorption).

[0009] Phthalophyllin may be further degraded into pyrophyllin due to the activity of plant enzymes during oilseed harvesting and storage, or due to processing conditions (i.e., heat) during oil refining (see "Behaviour of Chlorophyll Derivatives in Canola Oil Processing", JAOCS, Vol. 9 (Sept. 1993), pages 837-841). One possible mechanism is the enzymatic hydrolysis of the methyl ester bond on the carbon ring of pheophyll, followed by the non-enzymatic conversion of the unstable intermediate to pyrophyllin. A 28-29 kDa enzyme called pheophytinase from *Chenopodium album* has been reported to catalyze a similar reaction with pheophytin to produce a phytol-free pyrophyllin derivative called pyrophyllin (see [link to article]). Figure 1Pyromethesin is less polar than pheophytic acid, resulting in lower water solubility and higher oil solubility compared to pheophytic acid.

[0010] Depending on processing conditions, pyrophyllin may be more abundant in vegetable oils during processing than both pheophytin and chlorophyll (see Table 9 in volume 2.2 of Bailey's Industrial Oil and Fat Products (2005), 6). th (See Table 9 in *Berley Industrial Oils and Fats Products*, 6th edition, Volume 2.2, edited by Fereidoon Shahidi, John Wiley & Sons, 2005). This is partly because magnesium is lost from chlorophyll during the harvesting and storage of plant material. The amount of pyrophyll-demagnesin in the oil can increase and may exceed the amount of pheophyll if prolonged heat treatment at 90°C or higher is used. Chlorophyll levels are also reduced by heating oil-containing seeds before pressing and extraction, as well as by degumming and alkali treatment of the oil during refining processes. Phospholipids in the oil have also been observed to complex with magnesium, thus reducing the amount of chlorophyll. Therefore, chlorophyll is a relatively minor contaminant compared to pyrophyll-demagnesin (and pheophyll) in many vegetable oils.

[0011] Each of the four chlorophyll derivatives (chlorophyll a and b, and pheophytin a and b) exists as a pair of epimers, which are numbered by carbon atom 13. 2 (According to the IUPAC system number, in) Figure 2 The stereostructure of H and COOCH3 surrounding the asterisk (marked with an asterisk) determines the chlorophyll a. Therefore, chlorophyll a exists as epimers of chlorophyll a and chlorophyll a′, and chlorophyll b contains forms b and b'. Similarly, pheophytin a contains epimers a and a', and pheophytin b contains forms b and b'. The apostrophe (') form has a stereostructure surrounding carbon 13. 2 The S-stereostructure of the atom, rather than the apostrophe form, has a ring around carbon-13. 2The R-stereostructure of the atom. Epimerization, for example, the conversion from the α form to the α' form and vice versa, can occur in some cases via common enols, as described in "Epimerization in the pheophytina / a' system," Chemistry Letters (1984), 1411-1414. In solution, there is usually an equilibrium state indicating the distribution of pheophytina compounds in the α and α forms, and this is often determined by physical parameters such as temperature, pH, and solvent.

[0012] Generally, enzymes typically act as stereospecific catalysts because they are active only for one stereoisomer. The preceding analysis showed that chlorophyllase has a high degree of stereospecificity, catalyzing only the hydrolysis of non-pith forms of chlorophyll compounds (see "The stereospecific interaction between chlorophylls and chlorophyllase" J. Biol. Chem. 267(31):22043-22047 (1992)).

[0013] In methods for removing chlorophyll and chlorophyll derivatives from vegetable oils using chlorophyllase-associated enzymes, the stereospecificity of the enzymes can pose challenges. Specifically, complete degradation of the chlorophyll component can be very difficult depending on the distribution and equilibrium of chlorophyll stereoisomers in the oil. For example, if a significant proportion of chlorophyll or chlorophyll derivatives are present in a thallium form, this portion of chlorophyll derivatives in the oil may resist enzymatic degradation. Furthermore, many enzymes exhibit significantly lower activity against pyrophyllene chlorophyll than, for example, against pheophytin.

[0014] This problem with existing methods Figure 3 As shown in the image. Figure 3This describes the epimerization of pheophytin a and its conversion to pyropheophytin. The pH of a water / crude vegetable oil mixture (containing approximately 1-2% water) is typically around 5.0 at approximately 60°C. Under these conditions, the epimer distribution of pheophytin a in crude soybean or rapeseed oil is typically approximately 70% pheophytin a (R-steriform) and 30% pheophytin a′ (S-steriform), and isomerization between these two epimers is slow. Furthermore, depending on the reaction conditions, a variable amount of pyropheophytin can be formed. If the enzyme used in the reaction is predominantly active only against pheophytin a, a significant proportion of chlorophyll derivatives present in the oil cannot be directly hydrolyzed by the enzyme without pH adjustment.

[0015] Therefore, improved methods are needed to remove chlorophyll and chlorophyll derivatives (such as pheophytin and pyropheophytin) from vegetable oils. Specifically, methods are needed to remove various forms of chlorophyll and chlorophyll derivatives from oils. Summary of the Invention

[0016] In one aspect, the present invention provides a method for processing vegetable oil, comprising the step of contacting the vegetable oil with an enzyme, wherein the enzyme is capable of hydrolyzing chlorophyll or an a' or b' stereoisomer of a chlorophyll derivative.

[0017] In one embodiment, the stereoisomer of a' or b' includes chlorophyll a', pheophytin a', chlorophyll b', or pheophytin b'. Preferred stereoisomers are the a' stereoisomers of chlorophyll or chlorophyll derivatives, such as chlorophyll a' or pheophytin a'.

[0018] In one embodiment, the enzyme has an activity ratio of less than 10, less than 5, or less than 2 to the a' stereoisomer of chlorophyll or a chlorophyll derivative. In an alternative embodiment, the enzyme has an activity ratio of less than 10, less than 5, or less than 2 to the b' stereoisomer of chlorophyll or a chlorophyll derivative.

[0019] In one embodiment, after enzymatic treatment, the oil comprises, as a total of α and α' stereoisomers of chlorophyll or chlorophyll derivatives in the oil, at least 50% of the α stereoisomers of chlorophyll or chlorophyll derivatives. In an alternative embodiment, after enzymatic treatment, the oil comprises, as a total of β and β' stereoisomers of chlorophyll or chlorophyll derivatives in the oil, at least 50% of the β stereoisomers of chlorophyll or chlorophyll derivatives.

[0020] In another embodiment, the enzyme has an activity ratio of less than 10, less than 8, or less than 5 for pheophytin compared to pyrophyllin.

[0021] In another embodiment, the enzyme includes chlorophyllase, pheophytinase, and / or pyrophyllase activity, i.e., hydrolytic activity against chlorophyll, pheophytin, and / or pyrophyll.

[0022] In another embodiment, the enzyme is derived from Arabidopsis thaliana or castor bean (Ricinus communis). For example, the enzyme may comprise a polypeptide sequence as defined in SEQ ID NO:2 or SEQ ID NO:13, or a functional fragment or variant thereof.

[0023] Preferably, the enzyme comprises a polypeptide sequence having at least 75% sequence identity with SEQ ID NO:2 or SEQ ID NO:13 within a range of at least 50 amino acid residues. In one embodiment, the enzyme comprises a polypeptide having at least 90% sequence identity with SEQ ID NO:2. In another embodiment, the enzyme comprises a polypeptide having at least 90% sequence identity with SEQ ID NO:13.

[0024] In another aspect, the present invention provides vegetable oils that can be obtained according to the method of any one of the preceding claims.

[0025] In another aspect, the present invention provides the use of an enzyme capable of hydrolyzing chlorophyll or chlorophyll derivatives to remove the a' or b' stereoisomers of chlorophyll or chlorophyll derivatives from vegetable oils.

[0026] As described herein, enzymes exhibiting surprising hydrolytic activity against both chlorophyll derivatives in flaked and non-flaked forms have been identified. Furthermore, these enzymes also demonstrate enhanced hydrolytic activity against pyrophyllin compared to enzymes used in known methods. These enzymes can be advantageously used to enhance the removal of various forms of chlorophyll derivatives from vegetable oils. Attached Figure Description

[0027] Figure 1 This illustrates the reactions involving chlorophyll and its derivatives, as well as enzymes, used in this invention.

[0028] Figure 2 The image shows pheophytin a, with C-13 marked with an asterisk according to the IUPAC numbering system. 2 .

[0029] Figure 3 This demonstrates the epimerization of pheophytin a molecules and their conversion to pyropheophytin a.

[0030] Figure 4 The amino acid and nucleotide sequences are shown, which reveal the fusion of the chlorophyllase gene with a histidine tag and a thrombin site.

[0031] Figure 5 This diagram shows the expression vector pET28-TRI_CHL of Escherichia coli, which contains the TRI_CHL gene (database accession number BT009214) encoding a chlorophyllase from common wheat (Triticum aestivum).

[0032] Figure 6 The amino acid and nucleotide sequences showing the fusion of the chlorophyllase gene with the AprE signal sequence and the AGK sequence are displayed.

[0033] Figure 7 This diagram shows the expression vector pBN-TRI_CHL of Bacillus subtilis, which contains the TRI_CHL gene (database accession number BT009214) encoding chlorophyllase from common wheat.

[0034] Figure 8 The amino acid and nucleotide sequences showing the direct fusion of the chlorophyllase gene with the AprE promoter are displayed.

[0035] Figure 9 This diagram shows the expression vector pBN-Spe-TRI_CHL of Bacillus subtilis, which contains the TRI_CHL gene (database accession number BT009214) encoding chlorophyllase from common wheat.

[0036] Figure 10 The amino acid and nucleotide sequences showing the fusion of the chlorophyllase gene with the Cel A signal sequence are displayed.

[0037] Figure 11 This diagram shows the expression vector pKB-TRI_CHL of *Streptomyces lividans*, which contains the TRI_CHL gene (database accession number BT009214) encoding a chlorophyllase derived from common wheat.

[0038] Figure 12 The amino acid sequence of chlorophyllase from Arabidopsis thaliana is shown (SEQ ID NO:1).

[0039] Figure 13 The amino acid sequence of Arabidopsis chlorophyllase is shown (SEQ ID NO:2).

[0040] Figure 14 The amino acid sequence of chlorophyllase from sweet orange (Citrus sinensis) is shown (SEQ ID NO:3).

[0041] Figure 15 The amino acid sequence of common wheat chlorophyllase is shown (SEQ ID NO:4).

[0042] Figure 16 The amino acid sequence of common wheat chlorophyllase is shown (SEQ ID NO:5).

[0043] Figure 17 The amino acid sequence of chlorophyllase from Brassica oleracea is shown (SEQ ID NO:6).

[0044] Figure 18 The amino acid sequence of chlorophyllase from cabbage is shown (SEQ ID NO:7).

[0045] Figure 19 The amino acid sequence of chlorophyllase from cabbage is shown (SEQ ID NO:8).

[0046] Figure 20 The amino acid sequence of chlorophyllase from Zea Mays is shown (SEQ ID NO:9).

[0047] Figure 21 The amino acid sequence of corn chlorophyllase is shown (SEQ ID NO:10).

[0048] Figure 22 The amino acid sequence of chlorophyllase from moso bamboo (Phyllostachys edulis) is shown (SEQ ID NO:11).

[0049] Figure 23 The amino acid sequence of quinoa chlorophyllase is shown (SEQ ID NO:12).

[0050] Figure 24 The amino acid sequence of castor chlorophyllase is shown (SEQ ID NO:13).

[0051] Figure 25 The amino acid sequence of soybean (Glycine max) chlorophyllase is shown (SEQ ID NO:14).

[0052] Figure 26 The amino acid sequence of chlorophyllase from Ginkgo biloba is shown (SEQ ID NO:15).

[0053] Figure 27 The amino acid sequence of chlorophyllase from the money tree (Pachira macrocarpa) is shown (SEQ ID NO:16).

[0054] Figure 28 The amino acid sequence of chlorophyllase from Populus trichocarpa is shown (SEQ ID NO:17).

[0055] Figure 29 The amino acid sequence of chlorophyllase from sorghum bicolor is shown (SEQ ID NO:18).

[0056] Figure 30 The amino acid sequence of sorghum chlorophyllase is shown (SEQ ID NO:19).

[0057] Figure 31 The amino acid sequence of grape (Vitis vinifera) chlorophyllase is shown (SEQ ID NO:20).

[0058] Figure 32 The amino acid sequence of chlorophyllase from Physcomitrellapatens is shown (SEQ ID NO:21).

[0059] Figure 33 The amino acid sequence of chlorophyllase from Aquilegia is shown (SEQ ID NO:22).

[0060] Figure 34 The amino acid sequence of chlorophyllase from Brachypodium distachyon is shown (SEQ ID NO:23).

[0061] Figure 35 The amino acid sequence of chlorophyllase from Medicago truncatula is shown (SEQ ID NO:24).

[0062] Figure 36 The amino acid sequence of chlorophyllase from Piperbetle leaves is shown (SEQ ID NO:25).

[0063] Figure 37 The amino acid sequence of chlorophyllase from Lotus japonicus is shown (SEQ ID NO:26).

[0064] Figure 38 The amino acid sequence of chlorophyllase from indica rice (Oryza sativa Indica) is shown (SEQ ID NO:27).

[0065] Figure 39The amino acid sequence of chlorophyllase from japonica rice (Oryza sativa Japonica) is shown (SEQ ID NO:28).

[0066] Figure 40 The amino acid sequence of chlorophyllase from japonica rice is shown (SEQ ID NO:29).

[0067] Figure 41 The amino acid sequence of chlorophyllase from North American spruce (Picea sitchensis) is shown (SEQ ID NO:30).

[0068] Figure 42 The amino acid sequence of Chlamydomonas chlorophyllase is shown (SEQ ID NO:31).

[0069] Figure 43 This displays a pedigree of plant chlorophyllases and the Chlamydomonas chlorophyllase (CHL_CHL) described herein.

[0070] Figure 44 Western blot analysis of E. coli extracts containing recombinant chlorophyllases derived from various species is shown. Lane 1: BAM_CHL CoRe 112. Lane 2: CIT_CHL CoRe 113A. Lane 3: ARA_CHL CoRe 114A. Lane 4: CB_CHL CoRe 127. Lane 5: GlyMax_CHL CoRe 133. Lane 6: Sor_CHL CoRe 134. Lane 7: ARA_CHL2 CoRe 135. Lane 8: BRA_CHL1 CoRe 136. The definitions of the source species of the enzymes corresponding to the above abbreviations are shown in Tables 1 and 2 below.

[0071] Figure 45 Western blot analysis of E. coli extracts containing recombinant chlorophyllases derived from various species is shown. Lane 1: SORG_CHL CoRe 137A. Lane 2: TRI_CHL2 CoRe 138A. Lane 3: ZEA_CHL2 CoRe 139. Lane 4: TRI_CHL CoRe 20. Lane 5: BRACH_CHL CoRe 156. Lane 6: PIP_CHL CoRe 158. Lane 7: PICEA_CHL CoRe 163. Lane 8: Vector control. The definitions of the source species of the enzymes corresponding to the above abbreviations are shown in Tables 1 and 2 below.

[0072] Figure 46The activity of recombinant enzymes derived from various species for pheophytin a is shown, expressed as the total pheophytin a (pheophytin a+a') level in ppm at each time point after treatment with each enzyme.

[0073] Figure 47 The activity of recombinant enzymes derived from various species for pheophytin a is shown, expressed as pyrophyllin a levels in ppm at each time point after treatment with each enzyme.

[0074] Figure 48 The percentage of the α stereoisomer of pheophytin in oil samples treated with recombinases derived from various species is shown, based on the total amount of pheophytin a and pheophytin a' stereoisomers in the treated oil samples.

[0075] Figure 49 The HPLC chromatograms shown are obtained using absorbance detection (430 nm), with peak numbers indicating the substances associated with the following: 1 = chlorophyll b; 2 = chlorophyll a; 3 = neoxanthophyll; 3' = neoxanthophyll isomer; 4 = neopigment; 5 = zeaxanthin; 6 = lutein; 7 = zeaxanthin difuran; 8 = zeaxanthin; 8' = zeaxanthin isomer; 9 = zeaxanthin; 10 = lutein; 10' = lutein Isomers; 10” = lutein isomer; 11 = pheophytin b; 12 = pheophytin a; 13 = chlorophyll b; 13' = chlorophyll b'; 14 = chlorophyll a; 14' = chlorophyll a'; 15 = pheophytin b; 15' = pheophytin b'; 16 = β-carotene; 17 = pheophytin a; 17' = pheophytin a'; 18 = pyropheophytin b; 19 = pyropheophytin a.

[0076] Figure 50 A diagram showing a method for refining oil according to an embodiment of the present invention is shown.

[0077] Figure 51 The logarithm of substrate concentrations of pheophytic acid a and a' was shown as a function of ARA_CHL2 (Arabidopsis thaliana chlorophyllase) dosage after 1 / 2 hour. Detailed Implementation

[0078] In one aspect, the present invention relates to a method for treating vegetable oils. Typically, this method 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, in the presence of chlorophyll and / or chlorophyll derivatives as contaminants.

[0079] Chlorophyll and chlorophyll derivatives

[0080] The term "chlorophyll derivative" generally refers to compounds containing a porphyrin (dihydroporphyrin) ring and a chlorophyll group (tail), including magnesium-free chlorophyll-containing derivatives such as pheophytin and pyropheophytin. Chlorophyll and (chlorophyll-containing) chlorophyll derivatives are typically green due to the presence of the porphyrin (dihydroporphyrin) ring in the molecule. The loss of magnesium from the porphyrin ring means that pheophytin and pyropheophytin are more brownish than chlorophyll. Therefore, the presence of chlorophyll and chlorophyll derivatives in oil can give the oil an unsightly green, pale green, or brown color. In one embodiment, this method can be performed to remove or reduce the green or brown color present in the oil. Therefore, this method can be referred to as a bleaching or decolorizing method.

[0081] The enzymes used in this method hydrolyze chlorophyll and chlorophyll-containing derivatives to cleave the chlorophyll tail from the dihydroporphyrin ring. Hydrolysis of chlorophyll and chlorophyll derivatives typically yields compounds such as phytogenophytin, pheophytin, and pyropheophytin, which are chlorophyll-free derivatives. These compounds still contain a chromophorphyrin ring; phytogenophytin is green, while pheophytin and pyropheophytin are reddish-brown. In some embodiments, it may also be desirable to remove these chlorophyll-free derivatives and reduce the green / red / brown color of the oil. Therefore, in one embodiment of the invention, the method may further include the step of removing or reducing the level of chlorophyll-free chlorophyll derivatives in the oil. The method may involve bleaching or decolorizing to remove the green and / or red / brown color of the oil.

[0082] Chlorophyll or chlorophyll derivatives can be in form a or b. Therefore, as used herein, the term "chlorophyll" includes both chlorophyll a and chlorophyll b. Similarly, when referring to pheophytin, pyropheophytin, phytophytin, pheophytinic acid, and pyropheophytin, both forms a and b are covered.

[0083] As described herein, chlorophyll and chlorophyll derivatives can exist as a pair of epimers, which are numbered by carbon atom 13. 2 (According to the IUPAC system number, in) Figure 2 The stereochemical determination around the asterisk (') indicates that chlorophyll a exists as an epimery pair of chlorophyll a and chlorophyll a', and chlorophyll b comprises forms b and b'. Magnesium-containing chlorophyll a comprises epimers a and a', and magnesium-containing chlorophyll b comprises forms b and b'. The apostrophe (') form has a stereochemical determination around carbon 13. 2 The S-stereostructure of the atom, rather than the apostrophe form, has a ring around carbon-13. 2 The R-stereostructure of the atom. When used generally in this document, the term "chlorophyll and chlorophyll derivatives" includes both the apostrophe and non-apostrophe forms.

[0084] vegetable oil

[0085] This method can be used to treat any vegetable oil to remove undesirable contamination caused by chlorophyll and / or chlorophyll derivatives. The oil can be derived from any type of plant and from any part of the plant, including the whole plant, leaves, stems, flowers, roots, plant protoplasts, seeds, and plant cells and their identical offspring. Plant species from which the product can be treated by the method of this invention originates include higher plants, including angiosperms (monocotyledons and dicotyledons) and gymnosperms. It includes plants at multiple ploidy levels, including polyploid, diploid, haploid, and hemizygous states.

[0086] In a preferred embodiment, the oil may include vegetable oils, including oils processed from oil-bearing seeds or fruits (e.g., seed oils such as canola oil and fruit oils such as palm oil). Suitable examples of oils include rice bran, soybean, canola, palm, olive, cottonseed, corn, palm kernel, coconut, peanut, sesame, moringa, or sunflower. The method of the present invention can be used in conjunction with methods for processing essential oils (e.g., those derived from fruit seed oils, such as grapeseed, apricot, borage, etc.). The method of the present invention can be used in conjunction with methods for processing high-phosphorus oils (e.g., soybean oil). Preferably, the oil is a crude vegetable oil.

[0087] Chlorophyll and chlorophyll derivatives in oil

[0088] Chlorophyll and / or chlorophyll derivatives (e.g., chlorophyll, pheophyll, and / or pyropheophyll) can exist in oil as contaminants or naturally as undesirable components in processed products. Chlorophyll and / or chlorophyll derivatives (e.g., chlorophyll, pheophyll, and / or pyropheophyll) can exist in oil at any level. Typically, chlorophyll, pheophyll, and / or pyropheophyll can exist in oil as natural contaminants at a rate of 0.001 to 1000 mg / kg (0.001 to 1000 ppm, 10) based on the total weight of the oil. -7 Up to 10 -1 The chlorophyll and / or chlorophyll derivatives may be present in the oil at concentrations 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.

[0089] Chlorophyll derivatives that do not contain phytol can also be present in the oil. For example, phytogen, pyromethechol, and / or pyromethechol can be present in the oil at any level. Typically, phytogen, pyromethechol, and / or pyromethechol can be present in the oil at 0.001 to 1000 mg / kg (0.001 to 1000 ppm, 10 mg / kg) based on the total weight of the oil before or after enzymatic treatment according to the method of the present invention. -7 Up to 10-1 The chlorophyll, pyrophyll, and / or pyrophyll may be present in the composition at concentrations 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.

[0090] Enzymes that hydrolyze chlorophyll or chlorophyll derivatives

[0091] The method of the present invention includes the step of contacting an oil with an enzyme capable of hydrolyzing chlorophyll or chlorophyll derivatives, particularly their skimmed stereoisomers (such as a' or b'). Generally, "hydrolyzing chlorophyll or chlorophyll derivatives" refers to hydrolyzing the ester bonds in chlorophyll or (chlorophyll-containing) chlorophyll derivatives, for example, by cleaving the chlorophyll groups from the dihydroporphyrin ring in chlorophyll or chlorophyll derivatives. Therefore, the enzyme typically has esterase or hydrolase activity. Preferably, the enzyme has esterase or hydrolase activity in the oil phase and optionally also in the aqueous phase.

[0092] Therefore, the enzyme can be, for example, chlorophyllase, pheophytin, or pyrophyllase. Preferably, the enzyme is capable of hydrolyzing at least one, at least two, or all three of chlorophyll, pheophytin, and pyrophyll. In a particularly preferred embodiment, the enzyme has chlorophyllase, pheophytin, and pyrophyllase activities. In other embodiments, two or more enzymes can be used in the method, each enzyme having different substrate specificity. For example, the method may include the combined use of two or three enzymes selected from chlorophyllase, pheophytin, and pyrophyllase.

[0093] Any polypeptide having the activity of hydrolyzing chlorophyll or chlorophyll derivatives (especially their apiculated stereoisomers) can be used as an enzyme in the process of this invention. The term "enzyme" is intended to encompass any polypeptide having hydrolytic activity against apiculated stereoisomers of chlorophyll or chlorophyll derivatives (such as a' or b'), including, for example, enzyme fragments. Any isolated, recombinant, synthetic, or chimeric (or a combination of synthetic and recombinant) polypeptide can be used.

[0094] In embodiments of the present invention, the enzyme is capable of hydrolyzing the a' or b' stereoisomers of chlorophyll or chlorophyll derivatives. That is, the enzyme has hydrolytic activity against the '' form of chlorophyll or chlorophyll derivatives. The '' designation refers to carbon atom number 13 in the chlorophyll or chlorophyll derivative. 2 The surrounding three-dimensional structure.

[0095] Therefore, in the embodiments of the present invention, the hydrolyzable chlorophyll or chlorophyll derivatives in the ablation form include chlorophyll a′, chlorophyll b′, pheophytin a′, and pheophytin b′. Preferably, the enzyme is capable of hydrolyzing at least the ablation form of class a chlorophyll derivatives, namely chlorophyll a′ or pheophytin a′.

[0096] Typically, the enzyme also exhibits hydrolytic activity towards the non-skimmed forms of chlorophyll or chlorophyll derivatives. The enzyme used in this invention may have reduced stereospecificity; that is, the enzyme used herein has lower specificity for the non-skimmed forms of chlorophyll or chlorophyll derivatives than other known chlorophyllases (e.g., common wheat chlorophyllase, see SEQ ID NO:4).

[0097] In one embodiment, the enzyme has an activity ratio of less than 100 to the non-piped form (e.g., a' or b') stereoisomer of chlorophyll or chlorophyll derivatives. Preferably, this activity ratio is less than 50, less than 10, less than 5, less than 3, less than 2, less than 1.5, less than 1, or less than 0.5. The term "activity ratio" refers to the relative activity of the enzyme to the piped form compared to the non-piped form under the same conditions. Therefore, the activity ratio can be determined by measuring (a) the hydrolytic activity of the enzyme to the non-piped stereoisomer and (b) the hydrolytic activity of the enzyme to the corresponding piped stereoisomer, and then dividing (a) by (b). Therefore, a low activity ratio indicates relatively high activity to the piped form.

[0098] Hydrolytic activity can be determined, for example, using the methods described below. Typically, the activity ratio can be determined under conditions that do not support epimerization (i.e., conversion between the skimmed and non-skimmed isomers). For example, the activity ratio can be determined by measuring the hydrolytic activity of the enzyme against the skimmed and non-skimmed isomers in crude oil containing greater than 0.5 ppm of pheophytin and about 2% water at pH 5.0 to 5.5. In one embodiment, the enzyme's hydrolytic activity against pheophytin a or pheophytin a' is calculated at half the initial substrate concentration (see Example 4).

[0099] In another embodiment, the enzyme has an activity ratio of less than 80, less than 50, less than 10, less than 8, less than 7, or less than 5 compared to pyromethechene. For example, the enzyme may have an activity ratio of 0.1 to 10, 1 to 10, or 1 to 5 for pheophytin. The activity ratio of pheophytin to pyromethechene can be calculated by determining the pheophytin and pyromethechene activities under the same conditions using the method described below, and dividing the pheophytin activity by the pyromethechene activity. The activity ratios within the above ranges can be determined for the corresponding species of pheophytin and pyromethechene, such as pheophytin a (including forms a and a') to pyromethechene a.

[0100] Enzyme (chlorophyllase, pheophytinase, or pyropheophytinase) activity assay

[0101] Hydrolytic activity against chlorophyll or chlorophyll derivatives (including their skimmed and non-skimmed forms) can be detected using any suitable analytical technique, such as the assays described herein. For example, fluorescence-based techniques can be used to detect hydrolytic activity. In a suitable assay, the peptide whose chlorophyll or chlorophyll derivative hydrolytic activity is to be tested is incubated in the presence of a substrate, and product or substrate levels are monitored by fluorescence measurement. Suitable substrates include chlorophyll, pheophytin, and / or pyropheophytin, including their a and b forms, as well as their skimmed and non-skimmed forms. Detectable products include phytochlorophyll, pheophytic acid, pyropheophytin, and / or phytol.

[0102] Determination methods for detecting the hydrolysis of chlorophyll or chlorophyll derivatives are disclosed, for example, in the following literature: Ali Khamessan et al. (1994), Journal of Chemical Technology & Biotechnology, 60(1), pages 73–81; Klein and Vishniac (1961), J. Biol. Chem. 236: 2544-2547; and Kiani et al. (2006), Analytical Biochemistry 353: 93–98.

[0103] Alternatively, suitable assays can be performed by HPLC detection and quantification based on substrate or product content after the addition of an enzyme, for example, based on the techniques described below. In one embodiment, the assay can be performed as described in Hornero-Mendez et al. (2005), Food Research International 38(8-9):1067-1072. In another embodiment, the following assay can be used:

[0104] Add 20 μl of 0.3 mM chlorophyll, pheophytin, or pyropheophytin dissolved in acetone to 170 μl mM HEPES at pH 7.0. Dissolve the enzyme in 50 mM HEPES at pH 7.0. Add 10 μl of enzyme solution to 190 μl of substrate solution to initiate the reaction and incubate at 40 °C for various time periods. Terminate the reaction by adding 350 μl of acetone. After centrifugation (2 min at 18,000 g), analyze the supernatant by HPLC to determine the amounts of (i) chlorophyll and phytochlorophyll, (ii) pheophytin and pheophytin, or (iii) pyropheophytin and pyropheophytin. Chlorophyll and chlorophyll derivatives in pheophytin-free and non-pheophytin forms can be distinguished by HPLC analysis, such as... Figure 49 As shown in the image.

[0105] One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes 1 micromolar of substrate (e.g., chlorophyll, pheophytin, or pyropheophytin) per minute at 40°C, for example, using the assay method described herein.

[0106] In some preferred embodiments, the enzymes used in this method have chlorophyllase, pheophytin, and / or pyropheophytin activity of at least 1000 U / g, at least 5000 U / g, at least 10000 U / g, or at least 50000 U / g, based on activity units per gram of purified enzyme, for example, as determined by the assay methods described herein. Preferably, the enzyme has hydrolytic activity against the stylomers of chlorophyll or chlorophyll derivatives (e.g., chlorophyll a', chlorophyll b', pheophytin a', or pheophytin b') in the p-form (e.g., a' or b') form, based on activity units per gram of purified enzyme.

[0107] In another embodiment, the hydrolytic activity against chlorophyll or chlorophyll derivatives can be determined using the method described in EP10159327.5.

[0108] Chlorophyllase

[0109] In one embodiment, the enzyme is capable of hydrolyzing at least one ephemeral form (e.g., a' or b') stereoisomer of chlorophyll. The hydrolysis of the ester bond of chlorophyll a' or b' can also be used in this method to generate a polypeptide of deplanted chlorophyll a' or b' and phytol. In one embodiment, the enzyme is a chlorophyllase classified according to the enzyme nomenclature classification system (EC 3.1.1.14). Isolated, recombinant, synthetic, or chimeric (a combination of synthetic and recombinant) polypeptides (e.g., enzymes or catalytic antibodies) can be used, see, for example, Marchler-Bauer (2003) Nucleic Acids Res. 31:383-387.

[0110] In one embodiment, the enzyme may be derived from Arabidopsis thaliana. For example, the enzyme may be a sequence containing SEQ ID NO:2 (see [link to SEQ ID NO:2]). Figure 13 ) of polypeptides.

[0111] In another embodiment, the chlorophyllase is derived from castor beans, such as castor beans. For example, the chlorophyllase may contain the sequence SEQ ID NO:13 (see [link to SEQ ID NO:13]). Figure 24 ) of polypeptides.

[0112] This document also provides enzymes comprising polypeptide sequences as defined in any of SEQ ID NO:1 to 31, as well as their functional fragments and variants, as described below.

[0113] Variations and fragments

[0114] Functional variants and fragments of known sequences of chlorophyll or chlorophyll derivatives in their apomorphic form can also be used in this invention. "Functional" means that the fragment or variant retains detectable hydrolytic activity against the stereoisomers of chlorophyll or chlorophyll derivatives in their apomorphic form (e.g., a' or b'). Typically, such variants and fragments show homology to the sequence of the source chlorophyllase, pheophytinase, or pyromethytinase, for example, to the amino acid sequence of the source chlorophyllase, pheophytinase, or pyromethytinase, such as SEQ ID:2 or SEQ ID NO:13, as if having at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity within a region of at least about 10, 20, 30, 50, 100, 200, 300, 500 or 1000 or more residues or over the entire length of the sequence.

[0115] The percentage of sequence identity can be determined using sequence comparison algorithms or by visual inspection. One such algorithm is the BLAST algorithm, such as BLAST version 2.2.2.

[0116] Other enzymes active against chlorophyll or chlorophyll derivatives suitable for the methods of this invention can be identified by determining the presence of conserved sequence motifs, such as those present in known chlorophyllase, pheophytinase, or pyropheophytinase sequences. For example, the motif GHSRG (SEQ ID NO: 32), which contains a serine active site, is highly conserved in chlorophyllase sequences. In some embodiments, the enzymes used in this invention may contain such sequences. Peptide sequences with suitable activity can be identified by searching genomic databases, such as the Microbiome Metagenomic Database (Joint Genome Institute of the U.S. Department of Energy (JGI-DOE, USA)), for the presence of these motifs.

[0117] Enzyme isolation and preparation

[0118] The enzymes suitable for use in this invention can be isolated from their natural sources or can be prepared, for example, using recombinant DNA technology. Nucleotide sequences encoding polypeptides having chlorophyllase, pheophytinase, and / or pyrophyllinase activities can be isolated or constructed and used to generate the corresponding polypeptides.

[0119] For example, a genomic DNA and / or cDNA library can be constructed using chromosomal DNA or messenger RNA from the organism that produced the polypeptide. If the amino acid sequence of the polypeptide is known, a labeled oligonucleotide probe can be synthesized and used to identify a polypeptide-encoding clone from a genomic library prepared from that organism. Alternatively, a labeled oligonucleotide probe containing a sequence homologous to the gene of another known polypeptide can be used to identify a polypeptide-encoding clone. In the latter case, less stringent hybridization and washing conditions are used.

[0120] Alternatively, clones encoding peptides can be identified by inserting a fragment of genomic DNA into an expression vector (such as a plasmid), converting the resulting genomic DNA library into enzyme-negative bacteria, and then inoculating the transformed bacteria onto an agar containing an enzyme that inhibits the peptide, thereby leading to the identification of clones expressing the peptide.

[0121] In another alternative, the nucleotide sequence encoding the polypeptide can be synthesized using established standard methods, such as the phosphoramidite method described by Beucage S.L. et al. (1981) Tetrahedron Letters 22, pp. 1859-1869 (Beucage S.L. et al., Tetrahedron Communications, Vol. 22, pp. 1859-1869), or the method described by Mattes et al. (1984) EMBO J. 3, pp. 801-805 (Matthes et al., Journal of the European Society for Molecular Biology, Vol. 3, pp. 801-805). In the phosphoramidite method, the oligonucleotide is synthesized, purified, renatured, ligated, and cloned into a suitable vector, for example, in an automated DNA synthesizer.

[0122] Nucleotide sequences can be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared according to standard techniques by ligating fragments of synthetic, genomic, or cDNA origin (as the case may be). Each ligated fragment corresponds to a portion of the entire nucleotide sequence. DNA sequences can also be prepared using specific primers via polymerase chain reaction (PCR), as described in, for example, US 4,683,202 or Saiki RK et al. (Science (1988) 239, pp. 487-491).

[0123] As used in this article, the term "nucleotide sequence" refers to an oligonucleotide or polynucleotide sequence, as well as its variants, homologs, fragments, and derivatives (such as portions thereof). This nucleotide sequence can be derived from the genome, from synthesis, or from recombination, and can be double-stranded or single-stranded, whether representing the sense or antisense strand.

[0124] Typically, nucleotide sequences encoding polypeptides with chlorophyllase, pheophytinase, and / or pyrophyllinase activities are prepared using recombinant DNA technology. However, in alternative embodiments of the present invention, all or part of the nucleotide sequence may be synthesized using chemical methods well known in the art (see Caruthers MH et al. (1980) NucAcids Res Symp Ser 215-23 (Caruthers MH et al., 1980, Proceedings of the Nucleic Acids Research Series, pp. 215-223) and Horn T et al. (1980) NucAcids Res Symp Ser 225-232 (Horn T et al., 1980, Proceedings of the Nucleic Acids Research Series, pp. 225-232)).

[0125] Modification of enzyme sequence

[0126] Once the nucleotide sequence encoding the enzyme has been isolated, or the putatively encoded nucleotide sequence has been identified, it may be necessary to modify the selected nucleotide sequence, for example, by mutating the sequence to prepare the enzyme of the present invention.

[0127] Mutations can be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation site. A suitable method is disclosed in Morinaga et al. (Biotechnology (1984) 2, pp. 646-649). Another method for introducing mutations into enzyme-encoding nucleotide sequences is described in Nelson and Long (Analytical Biochemistry (1989), 180, pp. 147-151).

[0128] Instead of site-directed mutagenesis as described above, mutations can be introduced randomly using commercially available kits, such as the GeneMorph PCR Mutagenesis Kit from Stratagenene or the Diversify PCR Random Mutagenesis Kit from Clontech. EP 0 583 265 mentions methods for optimizing PCR-based mutagenesis, which can also be used in combination with mutagenic DNA analogs (such as those described in EP 0 866 796). Error-prone PCR techniques are suitable for producing enzyme variants with preferred properties that hydrolyze chlorophyll and / or chlorophyll derivatives. WO0206457 mentions the molecular evolution of lipases.

[0129] A third method for obtaining new sequences involves fragmenting the dissimilar nucleotide sequences with any number of restriction endonucleases or enzymes such as DNase I, and then reassembling them to form the complete nucleotide sequence encoding the functional protein. Alternatively, one or more dissimilar nucleotide sequences can be used, and mutations can be introduced during the reassembly of the complete nucleotide sequence. DNA shuffling and family shuffling techniques are suitable for generating enzyme variants with preferred properties. Suitable methods for performing “shuffling” can be found in EP0752008, EP1138763, and EP1103606. Shuffling can also be combined with other forms of DNA mutagenesis, as described in US 6,180,406 and WO 01 / 34835.

[0130] Therefore, it is possible to generate multiple site-directed or random mutations in nucleotide sequences in vivo or in vitro, and subsequently screen for improved functionality of the encoded polypeptide using various methods. For example, molecular evolution can be performed using computer- and insilico- and exo-mediated recombination methods (see WO 00 / 58517, US 6,344,328, US 6,361,974), where the resulting variants retain very low homology to known enzymes or proteins. Such variants obtained thereby can have significant structural similarity to known chlorophyllases, pheophytinases, or pyrophyllinases, but with very low amino acid sequence homology.

[0131] Additionally, as a non-limiting example, a mutation or natural variant of a polynucleotide sequence can be recombinated with the wild type or other mutated or natural variants to produce a new variant. Such new variants can also be screened for improved functionality of the encoded polypeptide.

[0132] The application of the above-described and similar molecular evolution methods allows for the identification and selection of variants of the enzymes of the present invention with preferred properties without prior knowledge of the protein structure or function, and allows for the generation of unpredictable but advantageous mutations or variants. Numerous examples exist in the art of using molecular evolution to optimize or alter enzyme activity, including (but not limited to) one or more of the following: optimizing expression and / or activity in host cells or in vitro; increasing enzyme activity; altering substrate and / or product specificity; increasing or decreasing enzyme or structural stability; and altering enzyme activity / specificity under preferred environmental conditions (e.g., temperature, pH, substrate).

[0133] Those skilled in the art will understand that enzymes can be altered to improve their functionality using molecular evolution tools. Suitably, the nucleotide sequence encoding the enzyme used in this invention (e.g., chlorophyllase, pheophytinase, and / or pyropheophytinase) can encode a variant enzyme, i.e., when compared with the parent enzyme, the variant enzyme may contain at least one amino acid substitution, deletion, or insertion. The variant enzyme retains at least 1%, 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 99% identity with the parent enzyme. Suitable parent enzymes may include any enzyme having hydrolytic activity against chlorophyll and / or chlorophyll derivatives in their thallium form.

[0134] polypeptide sequence

[0135] The present invention also covers the use of amino acid sequences encoded by nucleotide sequences, said nucleotide sequences encoding enzymes used in any of the methods and / or uses of the present invention.

[0136] As used herein, the term "amino acid sequence" is synonymous with the terms "peptide" and / or "protein." In some cases, the term "amino acid sequence" is synonymous with the term "peptide." Amino acid sequences can be prepared / isolated from suitable sources, or can be prepared synthetically or using recombinant DNA techniques. Suitablely, amino acid sequences can be obtained from the peptides taught herein using standard techniques.

[0137] A suitable method for determining the amino acid sequence from an isolated peptide is as follows: The purified peptide can be lyophilized, and 100 μg of the lyophilized material can be 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 at 50 °C for 15 min under nitrogen atmosphere and with the addition of 5 μl of 45 mM dithiothreitol. After cooling to room temperature, 5 μl of 100 mM iodoacetamide can be added to derivatize the cysteine ​​residues under nitrogen atmosphere in the dark at room temperature for 15 min.

[0138] Add 135 μl of water and 5 μg of the endonuclease Lys-C dissolved in 5 μl of water to the above reaction mixture, then digest under nitrogen at 37°C for 24 hours. The resulting peptides can be separated by reversed-phase HPLC on a VYDAC C18 column (0.46 × 15 cm; 10 μm; The Separation Group, California, USA) using solvent A: 0.1% TFA dissolved in water and solvent B: 0.1% TFA dissolved in acetonitrile. Selected peptides can be further separated chromatographically on a Develosil C18 column using the same solvent system before N-terminal sequencing. Sequencing can be performed using an AppliedBiosystems 476A sequencer with pulsed liquid rapid cycling according to the manufacturer's instructions (Applied Biosystems, California, USA).

[0139] Sequence comparison

[0140] Here, the term "homology" means an entity that shares a certain degree of homology with the subject amino acid sequence and the subject nucleotide sequence. Here, the term "homology" may be equated with "identity." Homologous amino acid sequences and / or nucleotide sequences should provide and / or encode polypeptides that retain functional activity and / or enhance enzyme activity.

[0141] In the context of this specification, homologous sequences are intended to include amino acid sequences that share at least 75, 85, or 90% identity with the subject sequence, preferably at least 95 or 98%. Typically, homologs will contain the same active sites, etc., as the subject amino acid sequence. Although homology can also be considered based on similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of this specification, homology is preferably expressed based on sequence identity.

[0142] In the context of this specification, homologous sequences are intended to include nucleotide sequences that have at least 75, 85, or 90% identity, preferably at least 95 or 98%, with respect to the nucleotide sequence encoding the polypeptide of the present invention (the subject sequence). Typically, homologs will contain sequences encoding the same active site, etc., as the subject sequence. Although homology can also be considered based on similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of this specification, homology is preferably expressed based on sequence identity.

[0143] Homology comparisons can be performed visually, or more commonly, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage homology between two or more sequences. Percent homology can be calculated for continuous sequences, i.e., one sequence is aligned with other sequences, 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 "vacancy-free" alignment. Typically, this vacancy-free alignment is performed only over a relatively short range of residues.

[0144] While this method is simple and reliable, it fails to account for factors such as the misalignment of subsequent amino acid residues in an otherwise identical sequence pair, potentially leading to a significant reduction in % homology during global alignment. Therefore, most sequence alignment methods are designed to produce optimal alignments that account for possible insertions and deletions without excessively penalizing the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to attempt to maximize local homology.

[0145] However, these more sophisticated methods assign a "vacancy penalty" to every empty space in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few vacancies as possible (reflecting a higher correlation between the two compared sequences) will receive a higher score than one with many vacancies. Affine gap costs are commonly used, which impose a relatively high cost for the presence of a vacancy and a smaller penalty for each subsequent residue within that vacancy. This is the most commonly used vacancy scoring system. A high vacancy penalty will naturally produce the best alignment with fewer vacancies. Most alignment programs allow modification of the vacancy penalty; however, the default value is preferred when performing sequence alignments with such software.

[0146] The calculation of maximum % homology therefore first requires generating the optimal alignment while considering gap penalties. A suitable computer program for performing this alignment is Vector NTI Advance. TM 11 (Invitrogen Corp.). Other examples of software capable of sequence comparison include (but are not limited to): the BLAST package (see Ausubelet al 1999 Short Protocols in Molecular Biology, 4th Ed-Chapter 18) and FASTA (Altschul et al 1990 J. Mol. Biol. 403-410). Both BLAST and FASTA can be used for offline and online searches (see Ausubelet et al 1999, pp. 7-58 to 7-60). However, for some applications, Vector NTI Advance is used. TM Procedure 11 is preferred. A novel tool called BLAST 2Sequences can also be used to compare protein and nucleotide sequences (see FEMS Microbiol Lett 1999 174(2):247-50 (Federation of European Microbiological Societies Microbiology Newsletter, 1999, Vol. 174, No. 2, pp. 247-250) and FEMS Microbiol Lett 1999 177(1):187-8 (Federation of European Microbiological Societies Microbiology Newsletter, 1999, Vol. 177, No. 1, pp. 187-188)).

[0147] While the final homology percentage (%) can also be measured in terms of identity, the alignment process itself is not typically based on all-or-nothing pairwise comparisons. Instead, a scaled similarity score matrix is ​​usually used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. A common example of such a matrix is ​​the default matrix in the BLOSUM62 matrix-BLAST package. VectorNTI programs typically use either common defaults or a custom symbolic comparison table (if provided) (see the user manual for further details). For certain applications, VectorNTI advance is preferred. TM 11. Default values ​​for the package.

[0148] As an alternative, the homology percentage can be obtained using Vector NTI Advance. TM The multiple alignment features in 11 (Invitrogen Corp.) are used to calculate the features, which are based on an algorithm similar to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244 (Higgins DG and Sharp PM, 1988, Gene, Vol. 73, No. 1, pp. 237-244)). Once the software has produced the best alignment results, it is possible to calculate % homology, preferably % sequence identity. The software typically performs these calculations as part of sequence comparison and produces numerical results.

[0149] If a gap penalty is used to determine sequence identity, then the program's default parameters for pairwise alignment can preferably be used. For example, the following parameters are the current default parameters for BLAST2 pairwise alignment:

[0150]

[0151] In one embodiment, the sequence identity of the preferred nucleotide and / or amino acid sequences can be determined by BLAST2(blastn) using the scoring parameters set as defined above.

[0152] For the purposes of this invention, the degree of identity is based on the number of identical sequence elements. According to the invention, the degree of identity of amino acid sequences can be suitably determined using computer programs known in the art, such as Vector NTI Advance. TM 11 (Invitrogen Corp.) determined. For paired comparisons, the scoring parameter used is preferably BLOSUM62, where the penalty for vacancy presence is 11 and the penalty for vacancy extension is 1.

[0153] Suitably, the degree of identity of the nucleotide sequence is determined over 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, and preferably at least 100 consecutive nucleotides. Suitably, the degree of identity of the nucleotide sequence can be determined over the entire sequence.

[0154] Amino acid mutation

[0155] The sequence may also have insertions or substitutions of amino acid residues that produce silencing changes and result in functionally equivalent substances. Intentional amino acid substitutions can be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the residues, provided that the secondary binding activity of the substance is preserved. 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 and similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0156] Conservative substitutions can be performed, for example, according to the table below. Amino acids within the same block in the second column, and preferably amino acids within the same row in the third column, can be substituted for each other:

[0157]

[0158] This invention also covers possible homologous substitutions (in this document, substitution and replacement both refer to the exchange of existing amino acid residues for alternative residues), i.e., equivalent substitutions, such as basic to basic, acidic to acidic, polar to polar, etc. Non-homologous substitutions may also occur, i.e., the replacement of one class of residues with another class of residues, or involving the addition of non-natural amino acids, such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), ortholeucine ornithine (hereinafter referred to as O), pyridylalanine, thiophenealanine, naphthylalanine, and phenylglycine. Substitutions can also arise from non-natural amino acids.

[0159] Variant amino acid sequences may include suitable spacer groups that can be inserted between any two amino acid residues in the sequence. These spacer groups, in addition to amino acid spacers such as glycine or β-alanine residues, may include alkyl groups such as methyl, ethyl, or propyl groups. Another form of variation (involving one or more amino acid residues in a peptoid form) will be well known to those skilled in the art. To avoid confusion, "peptide-like form" is used to refer to variant amino acid residues in which the α-carbon substituent is located on the nitrogen atom of the residue rather than on the α-carbon. Methods for preparing peptides in peptide-like forms are known in the art, for example, Simon RJ et al., PNAS (1992) 89(20), 9367-9371 (Simon RJ et al., Proceedings of the National Academy of Sciences of the United States of America, 1992, Vol. 89, No. 20, pp. 9367-9371) and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134 (Horwell DC, Trends in Biotechnology, 1995, Vol. 13, No. 4, pp. 132-134).

[0160] nucleotide sequence

[0161] The nucleotide sequences used in this invention, or those encoding polypeptides having the specific properties defined herein, may contain synthetic or modified nucleotides. Various types of modifications to oligonucleotides are known in the art. This includes the addition of acridine or polylysine chains to the 3' and / or 5' ends of the molecule, along with methyl phosphonate and phosphate thioester backbones. For the purposes of this invention, it should be understood that the nucleotide sequences described herein can be modified by any method available in the art. Such modifications can be made to improve the in vivo activity or lifetime of the nucleotide sequence.

[0162] This invention also covers the use of nucleotide sequences complementary to the sequences discussed herein, or any derivatives, fragments, or derivatives thereof. If a sequence is complementary to a fragment, it can be used as a probe to identify similar coding sequences in other organisms, and so on.

[0163] Polynucleotides that are not 100% homologous to the sequences of this invention but fall within the scope of this invention can be obtained in a variety of ways. Other variants of the sequences described herein can be obtained, for example, by probing DNA libraries prepared from a range of individuals, such as individuals from different populations. Furthermore, other viral / bacterial or cellular homologs present in plant cells can be obtained, particularly cellular homologs, and fragments thereof will generally be able to selectively hybridize with the sequences shown in the sequence listing herein. Such sequences can be obtained by probing cDNA or genomic DNA libraries prepared from other plant species and probing such libraries under moderate to high stringency conditions with a probe containing all or part of any of the sequences in the accompanying sequence listing. Similar considerations are made for obtaining species homologs and allelic variants of the polypeptide or nucleotide sequences of this invention.

[0164] Variants and strains / species homologs can also be obtained using degenerate PCR, which will use primers designed to target sequences within variants and homologs that encode conserved amino acid sequences in the sequences of this invention. Conserved sequences can be predicted, for example, by comparing amino acid sequences from several variants / homologs. Sequence alignment can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used.

[0165] Primers used in degenerate PCR will contain one or more degenerate positions and will be used under less stringent conditions than those used to clone a sequence from a known sequence using single-sequence primers.

[0166] Alternatively, this polynucleotide can be obtained through site-directed mutagenesis of a characterized sequence. This can be useful, for example, in situations where a silent codon sequence alteration is needed to optimize the codon preference of the polynucleotide sequence expressed in a specific host cell. Further sequence alterations may be required to introduce restriction peptide recognition sites, or to change the properties or function of the peptide encoded by the polynucleotide.

[0167] The polynucleotides (nucleotide sequences) of the present invention can be used to generate primers (e.g., PCR primers), primers for variable amplification reactions, probes (e.g., probes labeled with radioactive or non-radioactive markers by conventional means), or the polynucleotides can be cloned into vectors. Such primers, probes, and other fragments will have a length of at least 15, preferably at least 20, for example at least 25, 30, or 40 nucleotides, and are also covered by the term "polynucleotides of the present invention" as used herein.

[0168] The polynucleotides, such as DNA polynucleotides, and probes according to the present invention can be generated by recombination, synthesis, or by any means available to those skilled in the art. They can also be cloned using standard techniques.

[0169] Typically, primers are produced synthetically, involving the stepwise preparation of the desired nucleic acid sequence, one nucleotide at a time. Techniques for automating this process are readily available in the field.

[0170] Longer polynucleotides are typically generated using recombinant methods, such as PCR (polymerase chain reaction) cloning. This involves preparing a pair of primers (e.g., primers of about 15 to 30 nucleotides) flanking the region of the enzyme sequence to be cloned, contacting the primers with mRNA or cDNA obtained from plant cells, performing a polymerase chain reaction under conditions that result in amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. Primers can be engineered to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.

[0171] Enzyme preparation and dosage

[0172] The enzymes used in the methods of this invention can be formulated or modified, for example, chemically modified, to improve oil solubility, stability, activity, or for immobilization. For example, the enzymes used in the methods of this invention can be formulated to be amphiphilic or more lipophilic. For example, the enzymes used in the methods of this invention can be embedded in, for example, liposomes or gels, such as alginate hydrogels or alginate microbeads or equivalents. The enzymes used in the methods of this invention can be formulated in micellar systems, such as ternary micelles (TMS) or reverse micelle systems (RMS) media. The enzymes used in the methods of this invention can be formulated as described in Yi (2002) J. of Molecular Catalysis B: Enzymatic, Vol. 19, pgs 319-325.

[0173] The enzymatic reactions of the method of the present invention, such as the step of contacting the oil with an enzyme capable of hydrolyzing chlorophyll or chlorophyll derivatives in their piezoresistive (e.g., a' or b') stereoisomers, can be carried out in one or more reaction vessels. In one aspect, the enzymatic reactions of the method of the present invention are carried out in a vegetable oil refining facility or plant.

[0174] The method of the present invention can be implemented using immobilized enzymes, such as immobilized chlorophyllase, pheophytinase, and / or pyrophyllinase. The enzyme can be immobilized on any organic or inorganic support. Exemplary inorganic supports include alumina, diatomaceous earth, Dowex-1-chloride, glass microbeads, and silica gel. Exemplary organic supports include DEAE-cellulose, alginate hydrogels, or alginate microbeads or equivalents. In various aspects of the present invention, enzyme immobilization can be optimized by physical adsorption onto an inorganic support. Enzymes used to implement the present invention can be immobilized in various media, including water, Tris-HCl buffer, and ternary micelle systems comprising Tris-HCl buffer, hexane, and a surfactant. Enzymes can be immobilized to any type of substrate, such as filters, fibers, columns, microbeads, colloids, gels, hydrogels, meshes, etc.

[0175] The enzyme can be added to the oil in any suitable amount. For example, the enzyme can be added in the 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 μmol of substrate (such as chlorophyll a or b, pheophytin a or b and / or pyropheophytin a or b, or their piezomers (such as a' or b') stereoisomers) at 40 °C under the assay conditions described in J. Biol. Chem. (1961) 236:2544-2547.

[0176] Enzyme reaction conditions

[0177] Typically, oil can be incubated (or mixed) with enzymes at a temperature between about 5°C and about 100°C, more preferably between 10°C and about 90°C, even more preferably between about 15°C and about 80°C, and even more preferably between about 20°C and about 75°C.

[0178] Phthaphyllin decomposes into pyrophyllin at high temperatures, which is generally less desirable because some chlorophyllases have lower activity towards pyrophyllin compared to pheophyphyllin. Furthermore, the chlorophyllase degradation product of pyrophyllin, pyrophyllin, is less water-soluble than pheophylic acid and therefore more difficult to remove from oil afterward. Enzymatic reaction rates increase at higher temperatures, but this is advantageous in minimizing the conversion of pheophyphyllin to pyrophyllin.

[0179] Based on the above description, in a particularly preferred embodiment, the oil and enzyme are incubated at a temperature below about 80°C, preferably below about 70°C, preferably about 68°C or lower, and preferably about 65°C or lower, to reduce the amount of conversion to pyrophyllin. However, to maintain a good reaction rate, it is preferable to keep the oil temperature above 50°C during incubation with the enzyme. Therefore, preferred temperature ranges for enzyme-oil incubation include about 50°C to below about 70°C, about 50°C to about 65°C, and about 55°C to about 65°C.

[0180] Preferably, when the enzyme is mixed with the oil, the oil temperature can be at the desired reaction temperature. The oil can be heated and / or cooled to the desired temperature before and / or during enzyme addition. Therefore, in one embodiment, it is contemplated that other steps in the method of the invention could be cooling and / or heating the oil.

[0181] The reaction time (i.e., the period of incubation of the enzyme with the oil, preferably accompanied by stirring) appropriately allows sufficient time for the hydrolysis of chlorophyll and chlorophyll derivatives (especially their piezoresistive (e.g., a' or b') stereoisomers) to form, for example, phytol and phytogenic chlorophyll, pheophytic chlorophyll, and / or pyropheophytic chlorophyll. For example, the reaction time can be at least about 1 minute, more preferably at least about 5 minutes, and even more preferably at least about 10 minutes. In some embodiments, the reaction time can be between about 15 minutes and about 6 hours, preferably between about 15 minutes and about 60 minutes, and preferably between about 30 minutes and about 120 minutes. In some embodiments, the reaction time can be up to 6 hours.

[0182] Preferably, the method is carried out at a pH between about 4.0 and about 10.0, more preferably between about 5.0 and about 10.0, even more preferably between about 6.0 and about 10.0, even more preferably between about 5.0 and about 7.0, even more preferably between about 5.0 and about 7.0, even more preferably between about 6.5 and about 7.0, for example at about 7.0 (i.e., neutral pH). In one embodiment, the method is preferably carried out between about 5.5 and 6.0.

[0183] When incubated (or mixed) with an enzyme, the water content in the oil may suitably be between about 0.5% and about 5%, more preferably between about 1% and about 3%, and even more preferably between about 1.5% and about 2%.

[0184] When using immobilized enzymes, the water activity of the immobilized enzymes can suitably be in the range of about 0.2 to about 0.98, preferably between about 0.4 and about 0.9, and more preferably between about 0.6 and about 0.8.

[0185] Oil separation

[0186] After the enzyme treatment step using the enzyme of the present invention, in one embodiment the treated liquid (e.g., oil) is separated by a suitable device (e.g., a centrifuge) to obtain the processed oil. After the enzyme treatment is completed, the processed oil may be washed with water or an organic or inorganic acid, such as acetic acid, citric acid, phosphoric acid, succinic acid, etc., or with a salt solution, if necessary.

[0187] Chlorophyll and / or chlorophyll derivative removal

[0188] The methods of the present invention involving enzyme treatment generally reduce the levels of chlorophyll and / or chlorophyll derivatives (particularly their skimmed forms, such as a' or b') stereoisomers in oil. For example, compared to the concentrations (by weight) of chlorophyll, pheophytin, and / or pyropheophytin present in the oil before treatment, the method can reduce the concentrations of chlorophyll a or b, pheophytin a or b, and / or pyropheophytin a or b, or their skimmed forms, such as a' or b', stereoisomers 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%. Therefore, in certain embodiments, based on the total weight of the oil, the concentration of chlorophyll and / or chlorophyll derivatives or their pied forms (such as a' or b') stereoisomers in the treated oil can 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.

[0189] If an enzyme with stereospecificity to the non-piperized form of chlorophyll or chlorophyll derivatives is used, the vegetable oil typically contains a reduced proportion of non-piperized stereoisomers after enzymatic treatment, compared to the total amount of residual non-piperized and piperized stereoisomers in the oil (see Example 3 and below). Figure 48 In contrast, in embodiments of the present invention, the enzymes used in the method of the present invention typically have reduced stereospecificity for non-pickled forms of chlorophyll and chlorophyll derivatives, meaning that the enzymes are typically capable of hydrolyzing not only the pickled forms but also the non-pickled forms. Therefore, after the treatment steps of the present invention, the proportion of residual non-pickled stereoisomers in the oil typically decreases less than when using stereospecific enzymes.

[0190] It has been found that, under normal circumstances, crude oils (such as crude soybean oil or rapeseed oil) can contain approximately 70% of non-piped stereoisomers (such as pheophytin a) and 30% of piped stereoisomers (such as pheophytin a'). In one embodiment, after enzymatic treatment, the oil contains at least 50% of non-piped stereoisomers (such as a and / or b) of chlorophyll or chlorophyll derivatives, based on the total amount of non-major (such as a and / or b) stereoisomers and piped stereoisomers (such as a' and / or a') of chlorophyll or chlorophyll derivatives in the oil. More preferably, the oil, after treatment, contains at least 55%, at least 60%, or at least 65% of non-piped stereoisomers of chlorophyll or chlorophyll derivatives.

[0191] In one embodiment, after enzymatic treatment, the oil contains at least 50%, at least 60%, or at least 65% pheophytin a based on the total amount of pheophytin a and pheophytin a' in the oil. In one embodiment, after enzymatic treatment, the oil contains at least 50%, at least 60%, or at least 65% pheophytin b based on the total amount of pheophytin b and pheophytin b' in the oil.

[0192] In these embodiments, typical conditions may be, for example, about 20°C to about 70°C (e.g., about 40°C or about 60°C), pH 5 to 8 (e.g., about pH 6.0 or about pH 7.0), and water content of 1 to 3% (e.g., about 2%). The treatment time may include, for example, at least 1 hour, preferably 2 hours or more, more preferably 4 hours or more.

[0193] Further processing steps

[0194] In typical vegetable oil processing methods, the oil is extracted in hexane, the crude vegetable oil is degummed, optionally neutralized with alkali, bleached (using, for example, clay adsorption, followed by discarding the clay), and deodorized to produce refined, bleached, and deodorized oil or RBD oil (see [link to relevant documentation]). Figure 50 The need for a degumming step depends on phosphorus content and other factors. The method of this invention can be used in conjunction with hexane-based extraction and / or enzyme-assisted oil extraction methods (see Journal of American Oil Chemists' Society (2006), 83(11), 973-979). Generally, the method of this invention can be used as described in Bailey's Industrial Oil and Fat Products (2005), 6... thThe oil processing steps are described in the book, Ed. by Fereidoon Shahidi, John Wiley & Sons (Berley Industrial Oils and Fats Products, 2005, 6th edition, edited by Fereidoon Shahidi, John Wiley & Sons).

[0195] In embodiments of the invention, the enzymatic reaction involving the use of an enzyme capable of hydrolyzing chlorophyll or chlorophyll derivatives can be carried out at different stages of the method, such as... Figure 50 As shown in the illustration. In a particular embodiment, the enzyme is contacted with the oil prior to the degumming step. In another embodiment, the enzyme may be contacted with the oil during the water degumming step. In yet another embodiment, the enzyme is contacted with the water-degummed oil, but this contact occurs before degumming is complete (e.g., before total degumming or alkali neutralization steps).

[0196] Following enzymatic treatment, further processing steps can help remove enzymatic hydrolysis products of chlorophyll and / or chlorophyll derivatives. For example, further processing steps can remove phytohemochlorophyll, pheophytic chlorophyll, pyropheophyll, and / or phytol.

[0197] Degumming

[0198] The degumming step in oil refining serves to separate phospholipids by adding water. The material precipitated through degumming is separated and further processed into a mixture of lecithin. Commercially available lecithin, such as soybean lecithin and sunflower lecithin, is a semi-solid or very viscous substance. They consist of a mixture of polar lipids (primarily phospholipids such as phosphatidylcholine) and trace components of triglycerides. As used herein, the term "degumming" refers to refining oil by removing phospholipids from it. In some embodiments, degumming may include the step of converting phospholipids (e.g., lecithin and phospholipids) into hydrated phospholipids.

[0199] The method of this invention can be used with any degumming process, particularly in embodiments where chlorophyll or chlorophyll derivative hydrolytic enzymes are contacted with oil before the degumming step. Suitable degumming methods include water degumming, ALCON oil degumming (e.g., for soybeans), Safinco degumming, "super degumming," ultrafiltration degumming, TOP degumming, combined degumming, dry degumming, and Enzymax. TMDegumming method. See, for example, U.S. Patents No. 6,355,693, No. 6,162,623, No. 6,103,505, No. 6,001,640, No. 5,558,781, No. 5,264,367, No. 5,558,781, No. 5,288,619, No. 5,264,367, No. 6,001,640, No. 6,376,689, WO 0229022, WO 98118912, etc. The various degumming processes incorporated by the method of this invention are described in Bockisch, M. (1998), Fats and Oils Handbook, The extraction of Vegetable Oils (Chapter 5), 345-445, AOCS Press, Champaign, Illinois.

[0200] Aqueous degumming typically refers to a step in which oil is incubated with water (e.g., 1 to 5% by weight) to remove phospholipids. Typically, aqueous degumming can be carried out at high temperatures, such as 50 to 90°C. The oil / water mixture may be stirred for, for example, 5 to 60 minutes to allow the phospholipids to separate into the aqueous phase, which is then removed from the oil.

[0201] Acid degumming can also be performed. For example, the oil can be contacted with an acid (e.g., a 0.1 to 0.5% solution of 50% citric acid or malic acid) at 60 to 70°C, mixed, contacted with 1 to 5% water, and cooled to 25 to 45°C.

[0202] Other suitable degumming steps used with the method of the present invention are described in WO 2006 / 008508. In one embodiment, the method includes contacting an oil with a chlorophyll or chlorophyll derivative hydrolase, followed by an enzymatic degumming step using an acyltransferase, as described in WO 2006 / 008508. Acyltransferases suitable for this method are also described in WO 2004 / 064537, WO 2004 / 064987 and WO 2009 / 024736. Any enzyme with acyltransferase activity (generally classified as EC2.3.1) can be used, particularly enzymes containing the amino acid sequence motif GDSX, where 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, the acyltransferase is a mutant Aeromonas salmonicida mature lipid acyltransferase (GCAT) with the Asn80Asp mutation.

[0203] In another embodiment, the method includes a degumming step using a phospholipase. Any enzyme having, for example, phospholipase A1 (EC3.1.1.32) or phospholipase A2 (EC3.1.1.4) activity, such as lecitase, can be used. Or pancreatic phospholipase A2 (Novozymes, Denmark). In one embodiment, the method includes contacting the oil with a chlorophyll or chlorophyll derivative hydrolase, followed by an enzymatic degumming step using a phospholipase, for example, using the degumming step described in US 5,264,367, EP 0622446, WO 00 / 32758 or Clausen (2001) “Enzymatic oil degumming by a novel microbial phospholipase,” Eur. J. Lipid Sci. Technol. 103:333-340 (European Journal of Lipid Science and Technology, Vol. 103, pp. 333-340).

[0204] In another embodiment, the degumming step may be an aqueous degumming step. In another embodiment, an enzymatic degumming step utilizing an enzyme (e.g., phospholipase C (IUB3.1.4.1)) may be used. Peptides with phospholipase C activity that may be used in the degumming step are disclosed, for example, in WO2008143679, WO2007092314, WO2007055735, WO2006009676, and WO03089620. Suitable phospholipase C for use in this invention is derived from Verenium Corporation, Cambridge, MA, USA.

[0205] Acid treatment / base neutralization

[0206] In some embodiments, an acid treatment / alkali neutralization step may be performed after water degumming to further reduce the phospholipid level in the oil. In another embodiment, a single degumming step including acid treatment / alkali neutralization may be performed. Such methods are generally referred to as full degumming or alkali refining.

[0207] It has been found that the acid treatment / base neutralization step is particularly effective for removing enzymatic hydrolysis products of chlorophyll, such as phloroglucinol, pheophytin, and pyrophyllin. Therefore, this step can be performed at any stage after the enzymatic treatment step. For example, this step may include the addition of an acid, such as phosphoric acid, followed by neutralization with a base, such as sodium hydroxide. After the acid / base neutralization treatment, compounds such as phloroglucinol, pheophytin, and pyrophyllin are extracted from the oil into the aqueous phase.

[0208] In such methods, the oil is typically first contacted 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 aid in the precipitation of phospholipids. The contact time can be, for example, 10 seconds to 30 minutes. Subsequently, an aqueous solution of an alkali (e.g., a 1 to 20% aqueous solution of sodium hydroxide) is added, for example, at a temperature of 50 to 90°C, and then incubated and mixed for 10 seconds to 30 minutes. The oil can then be heated to approximately 90°C, and the aqueous soap phase is separated from the oil by centrifugation.

[0209] Alternatively, further washing steps may be performed using, for example, sodium hydroxide or water.

[0210] Remove deplanting chlorophyll, pheophytin, and pyropheophytin.

[0211] The method of the present invention may optionally involve the removal of chlorophyll derivatives that do not contain chlorophyll, such as phytophyll, pheophytin, and pyrophyll (including their skimmed and unskimmed forms). Such products may be present in the composition, or may be naturally present as contaminants, or as unwanted components in the processed product, due to the enzymatic hydrolysis of chlorophyll or chlorophyll derivatives of the present invention. Pyrophyll may also be present in the composition due to the decomposition of pheophytin, which itself may be produced by the activity of an enzyme with pheophytinase activity on pheophytin, or pheophytin may be formed from phytophyll after the action of chlorophyllase on chlorophyll (see...). Figure 1 Processing conditions used for oil refining, especially heating, can promote the formation of the major component, pyrophyllin, for example, by promoting the conversion of pheophytin to pyrophyllin, which is then hydrolyzed to pyrophyllin.

[0212] In one embodiment, the method of the present invention reduces the content of phytochlorophyll, pheophytin, and / or pyrophyllin in the oil compared to either or both of the content before and after enzyme treatment. In some embodiments, the concentrations of phytochlorophyll, pheophytin, and / or pyrophyllin may increase after enzyme treatment. Typically, the method involves a step of removing phytochlorophyll, pheophytin, and / or pyrophyllin such that the concentration of such products is lower than the concentration after enzyme treatment. Preferably, the phytochlorophyll, pheophytin, and / or pyrophyllin generated by this enzymatic step are removed from the oil such that the final content of these products in the oil is lower than the content before enzyme treatment.

[0213] For example, compared to the concentration (by weight) of chlorophyll, pheophytin, and / or pyrophyllium chlorophyll present in the oil before or after the removal step of chlorophyll, pheophytin, and / or pyrophyllium chlorophyll (including their skimmed and non-skimmed forms), this method can reduce the concentration of chlorophyll, pheophytin, and / or pyrophyllium chlorophyll (including their skimmed and non-skimmed forms) 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%. Therefore, in certain embodiments, the concentrations of phytochlorophyll, pheophytin, and / or pyrophyllin in the oil after the removal step, based on the total weight of the composition (e.g., vegetable oil), 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.

[0214] The advantage of the method of the present invention is that reaction products such as phytochlorophyll, pheophytin, and / or pyropheophytin can be easily and readily removed from the oil via, for example, acid treatment / base neutralization steps. Therefore, in a preferred embodiment, chlorophyll and chlorophyll derivatives can be substantially removed from the oil without further processing steps, such as clay and / or silica treatment and deodorization (e.g., Figure 50 (Represented by the dashed box shown).

[0215] clay treatment

[0216] This method is particularly preferred as it does not include a clay treatment step. Avoiding the use of clay is advantageous for the reasons previously stated, especially in terms of reduced costs, reduced oil loss due to clay adhesion, and increased retention of beneficial compounds such as carotenoids and tocopherols.

[0217] In some embodiments, the method can be carried out without clay treatment steps and without deodorization steps, which results in an increase in the concentration of such useful compounds in the refined oil compared to methods involving clay treatment.

[0218] Silica treatment

[0219] While not always necessary, in some embodiments the method may include a silica treatment step, preferably following enzymatic treatment. For example, the method may include silica purification apparatus and methods known in the art, using adsorbent-free or low-adsorbent silica purification techniques, such as the TriSyl silica purification method (Grace Davison, Columbia, MD, USA) or SORBSIL R TMSilica (INEOS Silica, Joliet, IL, USA).

[0220] The silica treatment step can be used to remove any residual dephytochlorophyll, pheophytic acid, and / or pyrophyll or other polar components from the oil. For example, in some embodiments, the silica treatment step can be used as an alternative to the acid treatment / alkali neutralization (total degumming or alkali refining) step.

[0221] In one embodiment, the method includes a two-stage silica treatment, for example, comprising two silica treatment steps separated by a separation step in which silica is removed. 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.

[0222] Deodorize

[0223] In some embodiments, the method may include a deodorization step, typically as the final refining step in the method. In one embodiment, deodorization refers to steam distillation of the oil, which typically removes volatile odor and flavor compounds, tocopherols, sterols, steranols, 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 remove gases. Steam (e.g., 1-3% by weight) is blown through the oil, for example, for 15 to 120 minutes, to remove volatile compounds. The aqueous distillate may be collected.

[0224] In another embodiment, deodorization can be carried out using an inert gas (e.g., nitrogen) instead of steam. Therefore, the deodorization step may include bubble refining or bubbling with an inert gas (e.g., nitrogen), as described in AVTsiadi et al., “Nitrogen bubble refining of sunflower oil in shallow pools”, Journal of the American Oil Chemists' Society (2001), Volume 78(4), pages 381-385. The gas phase passing through the oil can be collected and optionally condensed, and / or volatile compounds derived therefrom can be extracted into the aqueous phase.

[0225] In some embodiments, the method of the present invention is carried out without clay treatment but includes a deodorization step. Useful compounds (e.g., carotenoids, sterols, steranols, and tocopherols) can be at least partially extracted from the oil into a distillate (e.g., an aqueous or nitrogen-containing distillate) obtained from the deodorization step. This distillate provides a valuable source of compounds such as carotenoids and tocopherols, which can be at least partially lost due to entrainment in methods including clay treatment.

[0226] The loss of tocopherol during bleaching depends on the bleaching conditions and the type of clay applied, but it has been reported that 20-40% of tocopherol can be removed during the bleaching step (K. Boki, M. Kubo, T. Wada, and T. Tamura, ibid., 69, 323 (1992)). There have been reports of tocopherol loss of up to 13% during the bleaching step in soybean oil processing (S. Ramamurthi, ARMc Curdy, and R.T. Tyler, in SSKoseoglu, KCRhee, and RF Wilson, eds., Proc. World Conf. Oilseed Edible Oils Process, vol. 1, AOCS Press, Champaign, Illinois, 1998, pp. 130–134).

[0227] Carotenoids may be removed from oils during the deodorization step in both clay-treated and non-clay-treated oils. Typically, the removal of colored carotenoids is controlled to produce an oil with a predetermined color within a specified range. The content of carotenoids and other volatile compounds in refined oils can be varied by altering the deodorization step. For example, in embodiments where a higher concentration of carotenoids in the oil is desired, the deodorization step can be carried out at a lower temperature (e.g., using steam at 200°C or lower). In such embodiments, the clay treatment step is particularly preferred to be avoided, as this results in a higher concentration of carotenoids in the refined oil.

[0228] Further enzyme treatment

[0229] In another aspect, the method of the present invention further includes the use of lipid acyltransferases, phospholipases, proteases, phosphatases, phytases, xylanases, amylases (e.g., α-amylase), glucans, polygalacturonases, galactosyllipases, cellulases, hemicellulases, pectins, and other plant cell wall degrading enzymes, as well as mixed enzyme products and cell lysates. Alternatively, the method of the present invention may be implemented in combination with other methods, such as enzymatic treatments (e.g., using glycoases, including cellulases, hemicellulases, and other degradation byproducts) or chemical methods (e.g., hexane extraction of soybean oil). In one embodiment, the method of the present invention may be implemented in combination with methods as defined in WO 2006031699.

[0230] The invention will now be further illustrated with reference to the following non-limiting examples.

[0231] Example 1

[0232] Identification and cloning of chlorophyllase

[0233] Using different methods (including BLAST), the NCBI database was searched, and several sequences were identified as chlorophyllases or sequences homologous to chlorophyllases. The names of the sequences, their origins, and their NCBI database access numbers are listed in Table 1.

[0234] Table 1: Chlorophylate enzymes used in this paper with the following accession numbers and names.

[0235] organism Database login number CHL name Arabidopsis AAG12547 ARA_CHL Arabidopsis NP_199199 ARA_CHL2 Sweet orange AAF59834 CIT_CHL common wheat BT009214 TRI_CHL common wheat BT008923 TRI_CHL2 cabbage AAN51935 BRA_CHL cabbage AAN51933 BRA_CHL1 cabbage AAN51934 Brass_CHL2 corn ACN32030 ZEA_CHL corn ACG44273 ZEA_CHL2 bamboo FP092915 BAM_CHL quinoa Q9LE89 CHE_CHL castor bean XP_002517075 CB_CHL soybeans BAF43704 GlyMax_CHL ginkgo AAP44978 Gin_CHL Money Tree ACO50429 PAC_CHL2 hairy poplar XP_002315752 POP_CHL sorghum XP_002459848 Sor_CHL sorghum XP_002445588 SORG_CHL Grape XP_002273926 Vitis_CHL Small bowl moss EDQ81786 PHYS_CHL Columbine AQU_CHL Two spikelets of short-stalked grass ADDN01001446 BRACH_CHL Tribulus terrestris alfalfa ACJ85964 MED_CHL betel leaves ABI96085 PIP_CHL Hundred Veins Root AK338339 LOTUS_CHL Indica rice EEC66959 ORYI_CHL Japonica rice NP_001064620 ORYJ1_CHL Japonica rice EEE50970 ORYJ2_CHL North American Spruce ACN40275 PICEA_CHL Chlamydomonas XP_001695577 CHL_CHL

[0236] Chlorophyllase sequence

[0237] The chlorophyllase sequences identified from the NCBI database are listed in Table 1, and the amino acid sequences are... Figures 12 to 42 The sequence is shown in (SEQ ID NO:1 to 31). Multiple sequence alignment of the selected chlorophyllase amino acid sequence reveals several conserved residues distributed throughout the sequence. The motif GHSRG (SEQ ID NO:32), containing the serine active site, is highly conserved. This alignment yields results such as... Figure 43 The genealogical tree shown.

[0238] Cloning in E. coli

[0239] Synthetic genes encoding the chlorophyllases shown in Table 1 were prepared. Each gene was codon-optimized for expression in *E. coli*. For cloning purposes, the genes were extended at the 5' end to include an NheI restriction site and at the 3' end to include an XhoI restriction site.

[0240] After digestion with NheI and XhoI restriction enzymes, the synthesized DNA was ligated into the *E. coli* expression vector pET-28a(+) (Novagen) digested with the same restriction enzymes. This vector includes a T7 promoter for controlling the expression of the inserted gene and a Lac operon. The chlorophyllase gene was fused with a histidine tag and a thrombin cleavage site in a frame-compliant manner for purification. Figure 4 The example shown). The resulting construct (example pET28-TRI_CHL in Figure 5 The cells were transformed into competent E. coli TOP10 cells (Invitrogen), and plasmids were isolated from the transformed colonies and sequenced for nucleotides to verify the correct sequence. All fusions were as expected.

[0241] Expression in Escherichia coli

[0242] For expression, the plasmid was transformed into the expression host *E. coli* BL21(DE3) (Novagen). Cells were cultured at 37°C in LB broth containing carbenicillin (50 mg / ml) until OD. 600 The concentration was 0.6-0.8. For induction, 1 mM IPTG was added to the culture and incubated at 25°C for another 20-24 hours, followed by cell harvesting by centrifugation. The recombinant chlorophyllase was released from the cell pellet by sonication, and cell debris was removed by centrifugation.

[0243] Cloning in Bacillus subtilis

[0244] To facilitate cloning and expression in Bacillus subtilis, the synthetic gene encoding chlorophyllase (Table 1) was codon-optimized for Bacillus subtilis. The gene was cloned into two different plasmids, one for intracellular expression and the other for secretion into culture medium (extracellular expression).

[0245] extracellular expression

[0246] The gene was extended at the 5' end to include a BssHII restriction site and a portion of the AprE signal sequence for fusion into the AprE signal sequence within the read frame, as well as a sequence encoding the amino acid AGK, thereby facilitating signal sequence excision. The gene was extended at the 3' end with a PacI restriction site. The BssHII and PacI-digested gene was ligated into the Bacillus subtilis expression vector pBN, digested with the same restriction enzyme. The pBN vector contains the AprE promoter and the AprE signal sequence. An example of the resulting fusion of the chlorophyllase gene with the AprE signal sequence is shown in [reference needed]. Figure 6 The final constructor (example pBN-TRI_CHL) will be displayed in [the image / file]. Figure 7The cells were transformed into competent E. coli TOP10 cells (Invitrogen), and plasmids were isolated from the transformed colonies and sequenced for nucleotides to verify the correct sequence. All fusions were as expected.

[0247] For expression, the plasmid was transformed into the expression host Bacillus subtilis BG6002. Cells were cultured at 33°C in Grant's Type II medium for 68 hours. Recombinant chlorophyllase was isolated from the medium after centrifugation to precipitate the cells.

[0248] Intracellular expression

[0249] The gene was extended at the 5' end to include a SpeI restriction site, allowing direct fusion of the gene with the AprE promoter in the Bacillus subtilis expression vector pBN, which does not possess the AprE signaling sequence. The gene was extended at the 3' end with a HindIII restriction site. The fusion of the chlorophyllase gene with the AprE promoter was performed... Figure 8 The result is shown in [the image / image]. The resulting construct (example pBN-Spe-TRI_CHL) is displayed in [the image / image]. Figure 9 The cells were transformed into competent E. coli TOP10 cells (Invitrogen), and plasmids were isolated from the transformed colonies and sequenced for nucleotides to verify the correct sequence. All fusions were as expected.

[0250] For expression, the plasmid was transformed into the expression host Bacillus subtilis BG6002. Cells were cultured at 33°C in Grant's II medium for 68 hours. Recombinant chlorophyllase was released from the culture by treatment with 1 mg / ml lysozyme at 30°C for 1 hour. Cell debris was removed by centrifugation, and chlorophyllase was recovered from the supernatant.

[0251] Cloning in *Streptomyces lividus*

[0252] To clone and express *Streptomyces pulvinatus*, the synthetic gene encoding chlorophyllase (Table 1) was codon-optimized for *Streptomyces pulvinatus*. For cloning purposes, the gene was extended at the 5' end to include the NheI restriction site and a portion of the CelA signal sequence for fusion within the read frame with the CelA signal sequence. The 3' end was extended to include the BamHI restriction site. The fusion of the chlorophyllase gene (TRI_CHL) with the CelA signal sequence was performed... Figure 10 The resulting construct (example pKB-TRI_CHL) is displayed in [the image / image / description]. Figure 11The cells were transformed into competent E. coli TOP10 cells (Invitrogen), and plasmids were isolated from the transformed colonies and sequenced for nucleotides to verify the correct sequence. All fusions were as expected.

[0253] Expression in Streptomyces pulmonale

[0254] For expression, the plasmid was transformed into protoplasts of the host strain *Streptomyces pulvinatus* g3s3. Cells were pre-cultured at 30°C in TSG medium supplemented with thiostreptin for 48 hours. The pre-culture was then diluted 10-fold in Strept Pdxn2 modified medium and incubated at 30°C for 96 hours. Recombinant chlorophyllase was isolated from the medium after centrifugation to precipitate the cells.

[0255] Example 2

[0256] Chlorophyllase activity

[0257] Multiple chlorophyllases were identified and expressed in *E. coli* through genome mining as described above. Extracts from *E. coli* containing plasmids containing chlorophyllase genes were analyzed to determine pheophytinase activity. The analytical assays can be performed as described in EP10159327.5. Alternatively, pheophytinase activity can be determined by methods described above (e.g., HPLC-based methods). The results are shown in Table 2.

[0258] Phthaphyllinase activity can be determined by hydrolyzing pheophytin a in a reaction buffer and then performing a fluorescence assay on the resulting pheophytic acid a. This assay may also be suitable for using pyrophyllin as a substrate. 1 U of enzyme activity is defined as the hydrolysis of 1 μmol of pheophytin a or pyrophyllin per minute at 40 °C.

[0259] Table 2: Phosphochlorophyllase activity of enzymes

[0260] enzymes enzyme Activity U / mL BAM_CHL CoRe112 0,32 CIT_CHL CoRe113-A 0,25 ARA_CHL CoRe114-A 5,19 CB_CHL CoRe127 0,10 GlyMax_CHL Core133 0,010 Sor_CHL Core134 6,14 ARA_CHL2 Core135 0,94 BRA_CHL1 Core136 1,21 SORG_CHL CoRe137-A 0,78 TRI_CHL2 Core138-A 0,19 ZEA_CHL2 Core139 0,03 TRI_CHL CoRe20 0,18 BRACH_CHL CoRe156 1,50 PIP_CHL CoRe158 0,01 PICEA_CHL CoRe163 0,05 Comparison empty carrier 0,000

[0261] The enzymes described in Table 2 were analyzed by Western blot analysis using the primary antibody generated in rabbits against purified TRI_CHL. Figure 44 and 45 The table shows the reactions of all enzymes from Table 2 with the generated antibodies.

[0262] Example 3

[0263] Hydrolysis of chlorophyll derivatives in vegetable oils

[0264] The ability of some enzymes to degrade chlorophyll components in oil systems was tested. The formulations are shown in Table 3. Crude rapeseed oil was weighed in a Wheaton glass and heated to 60°C with magnetic stirring. Water and enzymes were added. The sample was treated with high-shear mixing for 20 seconds, followed by incubation at 60°C with magnetic stirring. Samples were removed after reaction times of 0.5, 2, and 4 hours. The samples were centrifuged and analyzed by HPLC-MS.

[0265] Table 3: Formulations for testing chlorophyllase in oil systems

[0266]

[0267] Figure 46 The total content of pheophytin a (pheophytin a+a′) is shown as determined by HPLC-MS. Figure 47 The degradation of pyrophyllin a was observed. All five candidate enzymes were able to degrade pheophytin and pheophytin, but the activity for pyrophyllin varied significantly among the five tested enzymes.

[0268] In oil samples treated with chlorophyllase, the distribution of the pheophytin stereoisomers a and a′ was also analyzed. Surprisingly, we found significant differences in this distribution depending on the enzyme applied (see [link to article]). Figure 48 For BRA_CHL1 and TRI_CHL, the percentage of pheophytin a decreased to approximately half of the initial content; however, ARA_CHL2 and CB_CHL exhibited distributions comparable to the control and initial contents. Maintaining the initial distribution of stereoisomers throughout the reaction was significantly advantageous, as it meant that the overall reaction rate was independent of the epimerization of pheophytin a′ to a. These findings also indicate that ARA_CHL2 and CB_CHL are effective against C-13... 2 The groups at that location are not very sensitive. Both enzymes also showed sensitivity to C-13. 2 Phosphorus chlorophyll with two hydrogen atoms exhibits much better activity (see [link]). Figure 47 ).

[0269] Substrate specificity in in vitro assays

[0270] The relative activities of the above enzymes for pheophytin and pyrophyllin were measured in an in vitro assay system, as described in, for example, EP10159327.5. Table 4 shows the ratio of pheophytin activity to pyrophyllin activity.

[0271] Table 4: Ratio of pheophytin activity to pyropheophytin activity

[0272] enzymes The activity ratio of pheophytin to pyropheophytin SORG_CHL 173 ARA_CHL2 4 CB_CHL 4 TRI_CHL 45

[0273] Clearly, SORG_CHL exhibits relatively low activity towards pyrophyllin compared to TRI_CHL. CB_CHL and ARA2_CHL show different substrate specificities, with a significant improvement towards pyrophyllin. These findings are consistent with... Figures 46 to 48 The contents shown are interconnected.

[0274] Example 4

[0275] Relative activities of chlorophyllase on pheophytin a and a'

[0276] The dose-response of chlorophyllase in crude rapeseed oil was tested according to the formulations in Table 5.

[0277] Table 5

[0278]

[0279] Samples were collected after 1 / 2, 2, and 4 hours of reaction time and analyzed by HPLC-MS. To compare the activities of different chlorophyllases against the two isomers, enzyme activities against the two isomers were calculated at half the initial substrate concentration. The natural logarithm of substrate concentration as a function of enzyme dose (U / g) was plotted, as shown below. Figure 51 The results show that Arabidopsis thaliana chlorophyllase (ARA_CHL2) is involved.

[0280] according to Figure 51 The curves in the figure show the enzyme activities for pheophytin a and a' calculated at half the initial substrate concentration, as shown in Table 6.

[0281] Table 6: Calculation of the activity of ARA-CHL2 against pheophytin a and pheophytin a'

[0282]

[0283] Different enzymes can be compared under the same conditions based on their activity at half the initial substrate concentration. Table 7 compares the results from two different chlorophyllases.

[0284] Table 7: Chlorophylate activity against pheophytin a and a' isomers in crude rapeseed oil

[0285] pheophytin_a' pheophytin_a Relative activity enzymes μg / U / h μg / U / h a' / a ARA_CHL2,CoRe135 574 605 0,95 CB_CHL,CoRe127-A 141 343 0,41

[0286] The results in Table 7 indicate that ARA_CHL2 exhibits nearly identical activity against the pheophytin a and a' isomers. This is consistent with the observation that the ratio between the two isomers remained unchanged during enzymatic degradation with ARA_CHL2 (see Table 7). Figure 48In contrast, CB_CHL also showed significant hydrolytic activity against pheophytin a'. Expressed as the ratio of activity against pheophytin a to that against pheophytin a' (i.e., the reciprocal shown in Table 7), ARA_CHL2 had an activity ratio of 1.05, while CB_CHL had an activity ratio of 2.44.

[0287] in conclusion

[0288] This invention identified 31 chlorophyllase sequences and cloned and expressed them in *Escherichia coli*, *Bacillus subtilis*, or *Streptomyces pulvinatus*. Based on expression in *E. coli*, pheophytinase activity was detected in nearly half of the identified chlorophyllases (Table 2), and all of these chlorophyllases reacted with antibodies against TRI_CHL. Figure 44 and 45 In protein extracts that do not have detectable pheophytinase activity, any expressed chlorophyllase cannot be detected based on Western blotting using antibodies generated against TRI_CHL.

[0289] When testing candidate chlorophyllases in oil applications, differences in specificity for pheophytin and pyrophyll substrates were found. ARA_CHL2 and CB_CHL showed significantly better activity for pyrophyll compared to other tested candidate enzymes. Figure 47 For both candidate enzymes, the ratio of pheophytin a to a′ was not significantly altered during incubation in the oil assay. For other candidate enzymes tested, this ratio decreased significantly during incubation. In in vitro assays using pheophytin and pyropheophytin as substrates, the improved activity of ARA_CHL2 and CB_CHL towards pyropheophytin was also measured in the oil assay (Table 4).

[0290] HPLC analysis

[0291] In the examples presented herein, chlorophyll derivatives can typically be quantified by HPLC analysis using the following method. HPLC analysis is performed using the method described in "Determination of chlorophylls and carotenoids by high-performance liquid chromatography during olive lactic fermentation," Journal of Chromatography, 585, 1991, pp. 259-266.

[0292] Phthaphyllin, pheophytin, pyrophyllin, and pyrophyllin were determined by HPLC connected to a diode array detector. The method used a C18 column packed with material, and chlorophyll was separated by gradient elution. Peaks were assigned using chlorophyll A and B standards from Sigma-Aldrich, for example, according to... Figure 49 The representative HPLC chromatogram shown is taken from Journal of Chromatography, 585, 1991, 259-266.

[0293] All publications mentioned in the foregoing description are incorporated herein by reference. Various modifications and variations of the methods and systems described herein will be apparent to those skilled in the art without departing from the spirit and scope of the invention. Although the invention has been described in conjunction with specific preferred embodiments, it should be understood that the invention protected by the claims should not be unduly limited to these specific embodiments. In fact, various modifications to the modes of carrying out the invention that will be apparent to those skilled in the art of biochemistry and biotechnology or related fields are intended to fall within the scope of the following claims. sequence list <110> Danisco <120> Processing methods <130> P042731WO <150> US 61 / 445665 <151> 2011-02-23 <160> 41 <170> PatentIn version 3.5 <210> 1 <211> 324 <212> PRT <213> Arabidopsis thaliana <400> 1 Met Ala Ala Ile Glu Asp Ser Pro Thr Phe Ser Ser Val Val Thr Pro 1 5 10 15 Ala Ala Phe Glu Ile Gly Ser Leu Pro Thr Thr Glu Ile Pro Val Asp 20 25 30 Pro Val Glu Asn Asp Ser Thr Ala Pro Pro Lys Pro Val Arg Ile Thr 35 40 45 Cys Pro Thr Val Ala Gly Thr Tyr Pro Val Val Leu Phe Phe His Gly 50 55 60 Phe Tyr Leu Arg Asn Tyr Phe Tyr Ser Asp Val Leu Asn His Ile Ala 65 70 75 80 Ser His Gly Tyr Ile Leu Val Ala Pro Gln Leu Cys Lys Leu Leu Pro 85 90 95 Pro Gly Gly Gln Val Glu Val Asp Asp Ala Gly Ser Val Ile Asn Trp 100 105 110 Ala Ser Glu Asn Leu Lys Ala His Leu Pro Thr Ser Val Asn Ala Asn 115 120 125 Gly Lys Tyr Thr Ser Leu Val Gly His Ser Arg Gly Gly Lys Thr Ala 130 135 140 Phe Ala Val Ala Leu Gly His Ala Ala Thr Leu Asp Pro Ser Ile Thr 145 150 155 160 Phe Ser Ala Leu Ile Gly Ile Asp Pro Val Ala Gly Thr Asn Lys Tyr 165 170 175 Ile Arg Thr Asp Pro His Ile Leu Thr Tyr Lys Pro Glu Ser Phe Glu 180 185 190 Leu Asp Ile Pro Val Ala Val Val Gly Thr Gly Leu Gly Pro Lys Trp 195 200 205 Asn Asn Val Met Pro Pro Cys Ala Pro Thr Asp Leu Asn His Glu Glu 210 215 220 Phe Tyr Lys Glu Cys Lys Ala Thr Lys Ala His Phe Val Ala Ala Asp 225 230 235 240 Tyr Gly His Met Asp Met Leu Asp Asp Asp Leu Pro Gly Phe Val Gly 245 250 255 Phe Met Ala Gly Cys Met Cys Lys Asn Gly Gln Arg Lys Lys Ser Glu 260 265 270 Met Arg Ser Phe Val Gly Gly Ile Val Val Ala Phe Leu Lys Tyr Ser 275 280 285 Leu Trp Gly Glu Lys Ala Glu Ile Arg Leu Ile Val Lys Asp Pro Ser 290 295 300 Val Ser Pro Ala Lys Leu Asp Pro Ser Pro Glu Leu Glu Glu Ala Ser 305 310 315 320 Gly Ile Phe Val <210> 2 <211> 318 <212> PRT <213> Arabidopsis thaliana <400> 2 Met Ser Ser Ser Ser Ser Arg Asn Ala Phe Glu Asp Gly Lys Tyr Lys 1 5 10 15 Ser Asn Leu Leu Thr Leu Asp Ser Ser Ser Arg Cys Cys Lys Ile Thr 20 25 30 Pro Ser Ser Arg Ala Ser Pro Ser Pro Pro Lys Gln Leu Leu Val Ala 35 40 45 Thr Pro Val Glu Glu Gly Asp Tyr Pro Val Val Met Leu Leu His Gly 50 55 60 Tyr Leu Leu Tyr Asn Ser Phe Tyr Ser Gln Leu Met Leu His Val Ser 65 70 75 80 Ser His Gly Phe Ile Leu Ile Ala Pro Gln Leu Tyr Ser Ile Ala Gly 85 90 95 Pro Asp Thr Met Asp Glu Ile Lys Ser Thr Ala Glu Ile Met Asp Trp 100 105 110 Leu Ser Val Gly Leu Asn His Phe Leu Pro Ala Gln Val Thr Pro Asn 115 120 125 Leu Ser Lys Phe Ala Leu Ser Gly His Ser Arg Gly Gly Lys Thr Ala 130 135 140 Phe Ala Val Ala Leu Lys Lys Phe Gly Tyr Ser Ser Asn Leu Lys Ile 145 150 155 160 Ser Thr Leu Ile Gly Ile Asp Pro Val Asp Gly Thr Gly Lys Gly Lys 165 170 175 Gln Thr Pro Pro Pro Val Leu Ala Tyr Leu Pro Asn Ser Phe Asp Leu 180 185 190 Asp Lys Thr Pro Ile Leu Val Ile Gly Ser Gly Leu Gly Glu Thr Ala 195 200 205 Arg Asn Pro Leu Phe Pro Pro Cys Ala Pro Pro Gly Val Asn His Arg 210 215 220 Glu Phe Phe Arg Glu Cys Gln Gly Pro Ala Trp His Phe Val Ala Lys 225 230 235 240 Asp Tyr Gly His Leu Asp Met Leu Asp Asp Asp Thr Lys Gly Ile Arg 245 250 255 Gly Lys Ser Ser Tyr Cys Leu Cys Lys Asn Gly Glu Glu Arg Arg Pro 260 265 270 Met Arg Arg Phe Val Gly Gly Leu Val Val Ser Phe Leu Lys Ala Tyr 275 280 285 Leu Glu Gly Asp Asp Arg Glu Leu Val Lys Ile Lys Asp Gly Cys His 290 295 300 Glu Asp Val Pro Val Glu Ile Gln Glu Phe Glu Val Ile Met 305 310 315 <210> 3 <211> 329 <212> PRT <213> Sweet orange (Citrus sinensis) <400> 3 Met Ala Ala Met Val Asp Ala Lys Pro Ala Ala Ser Val Gln Gly Thr 1 5 10 15 Pro Leu Leu Ala Thr Ala Thr Leu Pro Val Phe Thr Arg Gly Ile Tyr 20 25 30 Ser Thr Lys Arg Ile Thr Leu Glu Thr Ser Ser Pro Ser Ser Pro Pro 35 40 45 Pro Pro Lys Pro Leu Ile Ile Val Thr Pro Ala Gly Lys Gly Thr Phe 50 55 60 Asn Val Ile Leu Phe Leu His Gly Thr Ser Leu Ser Asn Lys Ser Tyr 65 70 75 80 Ser Lys Ile Phe Asp His Ile Ala Ser His Gly Phe Ile Val Val Ala 85 90 95 Pro Gln Leu Tyr Thr Ser Ile Pro Pro Pro Ser Ala Thr Asn Glu Leu 100 105 110 Asn Ser Ala Ala Glu Val Ala Glu Trp Leu Pro Gln Gly Leu Gln Gln 115 120 125 Asn Leu Pro Glu Asn Thr Glu Ala Asn Val Ser Leu Val Ala Val Met 130 135 140 Gly His Ser Arg Gly Gly Gln Thr Ala Phe Ala Leu Ser Leu Arg Tyr 145 150 155 160 Gly Phe Gly Ala Val Ile Gly Leu Asp Pro Val Ala Gly Thr Ser Lys 165 170 175 Thr Thr Gly Leu Asp Pro Ser Ile Leu Ser Phe Asp Ser Phe Asp Phe 180 185 190 Ser Ile Pro Val Thr Val Ile Gly Thr Gly Leu Gly Gly Val Ala Arg 195 200 205 Cys Ile Thr Ala Cys Ala Pro Glu Gly Ala Asn His Glu Glu Phe Phe 210 215 220 Asn Arg Cys Lys Asn Ser Ser Arg Ala His Phe Val Ala Thr Asp Tyr 225 230 235 240 Gly His Met Asp Ile Leu Asp Asp Asn Pro Ser Asp Val Lys Ser Trp 245 250 255 Ala Leu Ser Lys Tyr Phe Cys Lys Asn Gly Asn Glu Ser Arg Asp Pro 260 265 270 Met Arg Arg Cys Val Ser Gly Ile Val Val Ala Phe Leu Lys Asp Phe 275 280 285 Phe Tyr Gly Asp Ala Glu Asp Phe Arg Gln Ile Leu Lys Asp Pro Ser 290 295 300 Phe Ala Pro Ile Lys Leu Asp Ser Val Glu Tyr Ile Asp Ala Ser Ser 305 310 315 320 Met Leu Thr Thr Thr His Val Lys Val 325 <210> 4 <211> 319 <212> PRT <213> Wheat (Triticum aestivum) <400> 4 Met Ala Ala Ala Ala Pro Ala Glu Thr Met Asn Lys Ser Ala Ala Gly 1 5 10 15 Ala Glu Val Pro Glu Ala Phe Thr Ser Val Phe Gln Pro Gly Lys Leu 20 25 30 Ala Val Glu Ala Ile Gln Val Asp Glu Asn Ala Ala Pro Thr Pro Pro 35 40 45 Ile Pro Val Leu Ile Val Ala Pro Lys Asp Ala Gly Thr Tyr Pro Val 50 55 60 Ala Met Leu Leu His Gly Phe Phe Leu His Asn His Phe Tyr Glu His 65 70 75 80 Leu Leu Arg His Val Ala Ser His Gly Phe Ile Ile Val Ala Pro Gln 85 90 95 Phe Ser Ile Ser Ile Ile Pro Ser Gly Asp Ala Glu Asp Ile Ala Ala 100 105 110 Ala Ala Lys Val Ala Asp Trp Leu Pro Asp Gly Leu Pro Ser Val Leu 115 120 125 Pro Lys Gly Val Glu Pro Glu Leu Ser Lys Leu Ala Leu Ala Gly His 130 135 140 Ser Arg Gly Gly His Thr Ala Phe Ser Leu Ala Leu Gly His Ala Lys 145 150 155 160 Thr Gln Leu Thr Phe Ser Ala Leu Ile Gly Leu Asp Pro Val Ala Gly 165 170 175 Thr Gly Lys Ser Ser Gln Leu Gln Pro Lys Ile Leu Thr Tyr Glu Pro 180 185 190 Ser Ser Phe Gly Met Ala Met Pro Val Leu Val Ile Gly Thr Gly Leu 195 200 205 Gly Glu Glu Lys Lys Asn Ile Phe Phe Pro Pro Cys Ala Pro Lys Asp 210 215 220 Val Asn His Ala Glu Phe Tyr Arg Glu Cys Arg Pro Pro Cys Tyr Tyr 225 230 235 240 Phe Val Thr Lys Asp Tyr Gly His Leu Asp Met Leu Asp Asp Asp Ala 245 250 255 Pro Lys Phe Ile Thr Cys Val Cys Lys Asp Gly Asn Gly Cys Lys Gly 260 265 270 Lys Met Arg Arg Cys Val Ala Gly Ile Met Val Ala Phe Leu Asn Ala 275 280 285 Ala Leu Gly Glu Lys Asp Ala Asp Leu Glu Ala Ile Leu Arg Asp Pro 290 295 300 Ala Val Ala Pro Thr Thr Leu Asp Pro Val Glu His Arg Val Ala 305 310 315 <210> 5<^ <211> 323 <212> PRT <213> Wheat <400> 5 Met Ala Ala Met Ala Thr Thr Val Phe Gln Ala Gly Pro Met Glu Val 1 5 10 15 [[ID=X39]]Asp Val Lys His Val Asp Lys Ser Met Ile Pro Asn Leu Ala Arg Pro 20 25 30 Leu Met Val Val Ala Pro Lys Glu Thr Gly Ala Tyr Pro Val Ile Val 35 40 45 Phe Leu His Gly Trp Asn Met Leu Asn Ser Trp Tyr Glu Gln Leu Leu 50 55 60 Thr His Val Ala Ser His Gly Phe Ile Ala Val Ala Pro Gln Leu Tyr 65 70 75 80 Trp Met Val Ser Glu Pro Asp Ala Asp Asp Ile Asp Ala Thr Lys Arg 85 90 95 Ile Thr Asn Trp Leu Ala Asp His Asp Lys Gly Leu Ala His Val Leu 100 105 110 Lys Asp Val Leu Lys Leu Glu His Val Glu Pro Asp Leu Ser Lys Leu 115 120 125 Ala Leu Ala Gly His Ser Arg Gly Gly Gln Thr Ala Phe Ala Val Ala 130 135 140 Leu Gly Leu Gly Asp Ala Lys Thr Lys Leu Glu Leu Lys Phe Ser Ala 145 150 155 160 Leu Ile Gly Val Asp Pro Val Ala Gly Val Ser Arg Ala Gln Gln Leu 165 170 175 Glu Pro Lys Val Leu Thr Phe Glu Pro Asp Cys Leu Asp Val Gly Met 180 185 190 Pro Val Leu Val Met Gly Thr Gly Leu Gly Pro Lys His Ile Gly Gly 195 200 205 Phe Pro Cys Ala Pro Val Gly Val Asn His Ala Glu Phe Tyr Lys Glu 210 215 220 Cys Ala Pro Pro Arg Tyr His Leu Val Val Lys Asp Tyr Gly His Leu 225 230 235 240 Asp Met Leu Asp Asp Asn Val Pro Tyr Ile Ile Asn Asn Cys Met Cys 245 250 255 Met Arg Asn Gln His Asp Thr Lys Asp Leu Ala Arg Arg Thr Met Gly 260 265 270 Gly Ala Met Val Ala Phe Leu Arg Ala Lys Leu Arg Ile Asp Val Arg 275 280 285 Asp Leu Ile Ala Ile Tyr His Asn Pro Glu Ile Ala Pro Ala Val Leu 290 295 300 Asp Gln Val Asp Glu Phe Leu Pro Cys Phe Val Gly Arg Pro Asn Pro 305 310 315 320 Ser Ser Val <210> 6 <211> 213 <212> PRT <213> Brassica oleracea <400> 6 Met Ser Pro Ser Phe Leu Phe Phe Thr Leu Phe Leu Ile Lys Glu Met 1 5 10 15 Ser Ser Ser Ser Ala Asn Ser Phe Glu Asp Gly Lys Tyr Lys Thr 20 25 30 Asp Leu Leu Thr Val Gly Leu Ser Ser Cys Cys Trp Lys Lys Pro Ser 35 40 45 Ser Ser Pro Thr Pro Gln Ser Pro Pro Lys Arg Leu Leu Val Ala Thr 50 55 60 Pro Val Glu Glu Gly Glu Tyr Pro Val Val Met Leu Leu His Gly Tyr 65 70 75 80 Leu Leu Tyr Asn Ser Phe Tyr Ser Gln Leu Met Leu His Val Ser Ser 85 90 95 His Gly Phe Ile Val Ile Ala Pro Gln Leu Tyr Ser Ile Ala Gly Pro 100 105 110 Asp Thr Met Asp Glu Ile Lys Ser Thr Ala Glu Ile Ile Asp Trp Leu 115 120 125 Ser Val Gly Leu Asn His Phe Leu Pro Pro Gln Val Thr Pro Asn Leu 130 135 140 Ser Lys Phe Ala Leu Ser Gly His Ser Arg Gly Gly Lys Thr Ala Phe 145 150 155 160 Ala Leu Ala Leu Lys Lys Phe Gly Tyr Ser Ser Asp Leu Lys Ile Ser 165 170 175 Ala Leu Ile Gly Ile Asp Val Gly Thr Val Phe Trp Thr Asn Gly Tyr 180 185 190 Gly Gln Tyr Ser Gly Glu Phe Phe Glu Gln Phe Asp Cys Arg Asn Asp 195 200 205 Arg Ile Val Glu Ser 210 <210> 7 <211> 324 <212> PRT <213> Cabbage <400> 7 Met Ala Gly Lys Glu Asp Ser Glu Thr Phe Phe Ser Ala Ala Thr Pro 1 5 10 15 Leu Ala Phe Glu Leu Gly Ser Leu Pro Thr Thr Val Ile Pro Ala Asp 20 25 30 Pro Ser Ala Thr Asp Leu Thr Ala Pro Pro Lys Pro Val Ile Ile Thr 35 40 45 Ser Pro Thr Val Ala Gly Thr Tyr Pro Val Val Leu Phe Phe His Gly 50 55 60 Phe Tyr Leu Arg Asn Tyr Phe Tyr Ser Asp Val Ile Asn His Val Ala 65 70 75 80 Ser His Gly Tyr Ile Val Val Ala Pro Gln Leu Cys Lys Ile Leu Pro 85 90 95 Pro Gly Gly Gln Val Glu Val Asp Asp Ala Gly Lys Val Ile Asn Trp 100 105 110 Thr Ser Lys Asn Leu Lys Ala His Leu Pro Ser Ser Val Asn Ala Asn 115 120 125 Gly Asn Tyr Thr Ala Leu Val Gly His Ser Arg Gly Gly Lys Thr Ala 130 135 140 Phe Ala Val Ala Leu Gly His Ala Ala Thr Leu Asp Pro Ser Ile Lys 145 150 155 160 Phe Ser Ala Leu Val Gly Ile Asp Pro Val Ala Gly Ile Ser Lys Cys 165 170 175 Ile Arg Thr Asp Pro Glu Ile Leu Thr Tyr Lys Pro Glu Ser Phe Asp 180 185 190 Leu Asp Met Pro Val Ala Val Ile Gly Thr Gly Leu Gly Pro Lys Ser 195 200 205 Asn Met Leu Met Pro Pro Cys Ala Pro Ala Glu Val Asn His Glu Glu 210 215 220 Phe Tyr Ile Glu Cys Lys Ala Thr Lys Gly His Phe Val Ala Ala Asp 225 230 235 240 Tyr Gly His Met Asp Met Leu Asp Asp Asn Leu Pro Gly Phe Val Gly 245 250 255 Phe Met Ala Gly Cys Met Cys Lys Asn Gly Lys Arg Lys Lys Ser Glu 260 265 270 Met Arg Ser Phe Val Gly Gly Ile Val Val Ala Phe Leu Lys Tyr Ser 275 280 285 Ile Trp Gly Glu Met Ser Glu Ile Arg Gln Ile Leu Lys Asp Pro Ser 290 295 300 Val Ser Pro Ala Arg Leu Asp Pro Ser Pro Glu Leu Glu Glu Ala Ser 305 310 315 320 Gly Tyr Leu Val <210> 8 <211> 321 <212> PRT <213> Cabbage <400> 8 Met Ser Ser Ser Ser Ser Arg Asn Ala Phe Val Asp Gly Lys Tyr Lys 1 5 10 15 Pro Asp Leu Leu Thr Val Asp Leu Ala Ser Arg Cys Arg Cys Tyr Lys 20 25 30 Thr Thr Pro Ser Ser Ser Leu Thr Pro Pro Pro Pro Pro Lys Ser Leu 35 40 45 Leu Val Ala Thr Pro Val Glu Glu Gly Glu Tyr Pro Val Val Met Leu 50 55 60 Leu His Gly Tyr Leu Leu Tyr Asn Ser Phe Tyr Ser Gln Leu Met Leu 65 70 75 80 His Val Ser Ser Tyr Gly Phe Ile Val Ile Ala Pro Gln Leu Tyr Asn 85 90 95 Ile Ala Gly Pro Asp Thr Ile Asp Glu Ile Lys Ser Thr Ala Glu Ile 100 105 110 Ile Asp Trp Leu Ser Val Gly Leu Asn His Phe Leu Pro Pro Gln Val 115 120 125 Thr Pro Asn Leu Ser Lys Phe Ala Leu Thr Gly His Ser Arg Gly Gly 130 135 140 Lys Thr Ala Phe Ala Val Ala Leu Lys Lys Phe Gly Tyr Ser Ser Glu 145 150 155 160 Leu Lys Ile Ser Ala Ile Ile Gly Val Asp Pro Val Asp Gly Thr Gly 165 170 175 Lys Gly Lys Gln Thr Pro Pro Pro Val Leu Thr Tyr Glu Pro Asn Ser 180 185 190 Phe Asn Leu Glu Lys Met Pro Val Leu Val Ile Gly Ser Gly Leu Gly 195 200 205 Glu Leu Ala Arg Asn Pro Leu Phe Pro Pro Cys Ala Pro Thr Gly Val 210 215 220 Asn His Arg Glu Phe Phe Gln Glu Cys Gln Gly Pro Ala Trp His Phe 225 230 235 240 Val Ala Lys Asp Tyr Gly His Leu Asp Met Leu Asp Asp Asp Thr Lys 245 250 255 Gly Leu Arg Gly Lys Ser Ser Tyr Cys Leu Cys Lys Asn Gly Glu Glu 260 265 270 Arg Lys Pro Met Arg Arg Phe Ile Gly Gly Ile Val Val Ser Phe Leu 275 280 285 Met Ala Tyr Leu Glu Asp Asp Asp Cys Glu Leu Val Lys Ile Lys Ala 290 295 300 Gly Cys His Glu Gly Val Pro Val Glu Ile Gln Glu Phe Glu Val Lys 305 310 315 320 Lys <210> 9 <211> 333 <212> PRT <213> Zea mays <400> 9 Met Ala Ala Ser Pro Val Ala Ile Gly Thr Ala Val Phe Gln Arg Gly 1 5 10 15 Pro Leu Arg Val Glu Ala Arg His Val Asp Tyr Ser Gln Val Pro Ser 20 25 30 Val Pro Lys Pro Leu Met Val Val Ala Pro Thr Asp Ala Gly Val Tyr 35 40 45 Pro Val Ala Val Phe Leu His Gly Cys Asn Thr Val Asn Ser Trp Tyr 50 55 60 Glu Ser Leu Leu Ser His Val Ala Ser His Gly Phe Ile Ala Val Ala 65 70 75 80 Pro Gln Leu Tyr Cys Val Thr Leu Asn Met Asn Asp Leu Lys Asp Ile 85 90 95 Asp Ala Thr Arg Gln Val Thr Ala Trp Leu Ala Asp Lys Gln Gln Gly 100 105 110 Leu Ala His Val Leu Ala Asn Ile Leu Gln Leu His Gly Val Arg Pro 115 120 125 Asp Leu Ser Arg Leu Ala Leu Ala Gly His Ser Arg Gly Gly Asp Thr 130 135 140 Ala Phe Ala Val Ala Leu Gly Leu Gly Pro Ala Ala Ser Asp Asp Asp 145 150 155 160 Asp Asn Asn Ala Asp Ala Gly Thr Ser Pro Ala Ala Leu Pro Leu Lys 165 170 175 Phe Ser Ala Leu Ile Gly Val Asp Pro Val Ala Gly Leu Ser Lys Gln 180 185 190 Ala Gln Val Glu Pro Lys Val Leu Thr Phe Arg Pro Arg Ser Leu Asp 195 200 205 Pro Gly Met Pro Ala Leu Val Val Gly Thr Gly Leu Gly Pro Lys His 210 215 220 Val Gly Gly Pro Pro Cys Ala Pro Ala Gly Val Asn His Ala Glu Phe 225 230 235 240 Tyr Asp Glu Cys Ala Pro Pro Arg Tyr His Val Val Leu Arg Asp Tyr 245 250 255 Gly His Met Asp Met Leu Asp Asp Asp Gly Val Pro Tyr Val Ile Asn 260 265 270 Asn Cys Met Cys Met Arg Asn Thr Lys Asp Thr Lys Asp Leu Ala Arg 275 280 285 Arg Ala Ile Gly Gly Ala Val Val Ala Phe Leu Arg Ala Thr Leu Glu 290 295 300 Asp Asp Asp Glu Asp Leu Lys Val Val Leu Glu Asn Arg Pro Gly Leu 305 310 315 320 Ser Pro Ala Val Leu Asp Pro Val Gly His Asp Leu Ala 325 330 <210> 10 <211> 346 <212> PRT <213> Maize <400> 10 Met Asn Leu Ala Ser Ala Val Arg Val Phe Leu Ser Tyr Cys Leu Leu 1 5 10 15 Leu His Arg Trp Met Gly Ser Glu Gln Ala Gly Gly Val Phe Asp Gln 20 25 30 Gly Gly His Ser Val Ser Leu Thr Arg Leu Asp Glu Ala Arg Ala Pro 35 40 45 Pro Arg Cys Ala Val Gln Ser Ser Leu Ser Ser Ala Ala Ser Leu Pro 50 55 60 Pro Lys Pro Leu Leu Val Ala Ala Pro Arg Glu Thr Gly Glu Tyr Pro 65 70 75 80 Val Ile Leu Phe Leu His Gly Tyr Leu Ala Val Asn Ser Phe Tyr Ser 85 90 95 Gln Leu Phe Glu His Val Ala Ser His Gly Phe Ile Val Val Gly Pro 100 105 110 Gln Leu Tyr Thr Ile Ser Gly Ala Asp Thr Thr Glu Glu Ile Asn Ser 115 120 125 Ala Ala Ala Val Ile Asp Trp Leu Ala Thr Gly Leu Pro Ser Thr Leu 130 135 140 Pro Leu Gly Val Arg Ala Asp Leu Thr Lys Val Ser Ile Ser Gly His 145 150 155 160 Ser Arg Gly Gly Lys Val Ala Phe Ala Leu Ala Leu Gly His Ala Lys 165 170 175 Ala Lys Leu Ala Val Pro Leu Ala Ala Val Val Ala Val Asp Pro Val 180 185 190 Asp Gly Met Gly Val Gly Lys Gln Thr Pro Pro Pro Ile Leu Thr Gly 195 200 205 Arg His Gly Ser Leu His Val Gly Ala Pro Thr Met Val Ile Gly Thr 210 215 220 Gly Leu Gly Glu Leu Pro Arg Gly Ser Leu Leu Pro Pro Cys Ala Pro 225 230 235 240 Arg Gly Val Ser His Ala Ala Phe Tyr Asp Glu Leu Asp Gly Ala Ala 245 250 255 Pro Ala Cys His Leu Val Ala Arg Asp Tyr Gly His Thr Asp Met Met 260 265 270 Asp Asp Asp Thr Pro Gly Ala Arg Gly Met Leu Thr Arg Thr Ile Cys 275 280 285 Arg Ser Gly Gly Ala Arg Ala Pro Met Arg Arg Phe Val Ala Gly Ala 290 295 300 Thr Val Ala Phe Leu Lys Lys Trp Val Ala Gly Asp Ala Ala Ala Met 305 310 315 320 Asp Ser Ile Thr Ala Arg Pro Asp Gln Ala Pro Ile Ala Leu Ser Val 325 330 335 Val Glu Phe Gly Asp Glu Lys Ala Ile Ala 340 345 <210> 11 <211> 312 <212> PRT <213> Phyllostachys edulis <400> 11 Met Ala Ala Thr Ala Glu Ile Lys Ile Pro Ser Thr Glu Ala Leu Glu 1 5 10 15 Ala Val Thr Ser Val Phe Arg Pro Gly Lys Leu Ala Val Glu Leu Val 20 25 30 Pro Val Asp His Asn Ala Val Pro Thr Pro Pro Ile Pro Ile Leu Ile 35 40 45 Val Ala Pro Lys Asp Ala Gly Thr Tyr Pro Val Ala Met Leu Leu His 50 55 60 Gly Phe Phe Leu Gln Asn His Phe Tyr Glu His Leu Leu Lys His Val 65 70 75 80 Ala Ser His Gly Phe Ile Met Val Ala Pro Gln Phe His Ala Ile Cys 85 90 95 Thr Gly Glu Thr Glu Asp Ile Ala Ala Ala Ala Lys Val Thr Asp Trp 100 105 110 Leu Pro Glu Gly Leu Pro Ser Val Leu Leu Lys Gly Val Glu Ala Asp 115 120 125 Leu Ser Lys Leu Ala Leu Ala Gly His Ser Arg Gly Gly His Thr Ala 130 135 140 Phe Ser Leu Ala Leu Gly His Gly Lys Thr Asn Leu Asn Phe Ala Ala 145 150 155 160 Leu Ile Gly Leu Asp Pro Val Ala Gly Thr Gly Lys Ser Ser Gln Leu 165 170 175 Pro Pro Lys Ile Leu Thr Tyr Lys Pro Ser Ser Phe Asp Val Ala Met 180 185 190 Pro Val Leu Val Ile Gly Thr Gly Leu Gly Glu Glu Lys Lys Asn Val 195 200 205 Leu Phe Pro Pro Cys Ala Pro Lys Asp Val Asn His Arg Glu Phe Tyr 210 215 220 Tyr Glu Cys Lys Pro Pro Cys Tyr Tyr Phe Val Thr Lys Asp Tyr Gly 225 230 235 240 His Leu Asp Met Leu Asp Asp Asp Ala Pro Lys Phe Ile Thr Cys Leu 245 250 255 Cys Lys Asp Gly Asp Asn Cys Lys Asp Lys Met Arg Arg Ala Val Ala 260 265 270 Gly Ile Met Ile Ala Phe Leu Arg Ala Val Leu Asp Glu Lys Asp Gly 275 280 285 Asp Ile Lys Val Ile Leu Lys Asp Pro Gly Leu Ala Pro Val Thr Leu 290 295 300 Asp Pro Val Glu Cys Arg Leu Pro 305 310 <210> 12 <211> 347 <212> PRT <213> Chenopodium album <400> 12 Met Ala Lys Leu Leu Leu Leu Ile Phe Gly Val Phe Ile Phe Val Asn 1 5 10 15 Ser Gln Ala Gln Thr Phe Pro Thr Ile Leu Glu Lys His Asn Ser Glu 20 25 30 Lys Ile Thr Asp Val Phe His Lys Gly Asn Phe Gln Val Thr Asn Asn 35 40 45 Pro Ile Arg Val Lys Arg Tyr Glu Phe Ser Ala Pro Glu Pro Leu Ile 50 55 60 Ile Ile Ser Pro Lys Glu Ala Gly Val Tyr Pro Val Leu Leu Phe Ile 65 70 75 80 His Gly Thr Met Leu Ser Asn Glu Asp Tyr Ser Leu Phe Phe Asn Tyr 85 90 95 Ile Ala Ser His Gly Phe Ile Val Val Ala Pro Lys Leu Phe Arg Leu 100 105 110 Phe Pro Pro Lys Leu Pro Ser Gln Gln Asp Glu Ile Asp Met Ala Ala 115 120 125 Ser Val Ala Asn Trp Met Pro Leu Tyr Leu Gln Val Val Leu Gln Arg 130 135 140 Tyr Val Thr Gly Val Glu Gly Asp Leu Glu Lys Leu Ala Ile Ser Gly 145 150 155 160 His Ser Arg Gly Gly Lys Ser Ala Phe Ala Leu Ala Leu Gly Phe Ser 165 170 175 Asn Ile Lys Leu Asp Val Thr Phe Ser Ala Leu Ile Gly Val Asp Pro 180 185 190 Val Ala Gly Arg Ser Val Asp Asp Arg Thr Leu Pro His Val Leu Thr 195 200 205 Tyr Lys Pro Asn Ser Phe Asn Leu Ser Ile Pro Val Thr Val Ile Gly 210 215 220 Ser Gly Leu Gly Asn His Thr Ile Ser Cys Ala Pro Asn His Val Ser 225 230 235 240 His Gln Gln Phe Tyr Asp Glu Cys Lys Glu Asn Ser Ser His Phe Val 245 250 255 Ile Thr Lys Tyr Gly His Met Asp Met Leu Asn Glu Phe Arg Leu Ser 260 265 270 Pro Ile Ala Val Thr Met Ser Leu Met Cys Ala Gln Ser Phe Arg Pro 275 280 285 Lys Ala Thr Met Arg Arg Thr Leu Gly Gly Ile Met Val Ala Phe Leu 290 295 300 Asn Ala Tyr Phe Arg Asp Asp Gly Arg Gln Tyr Tyr Ala Ile Ile Ala 305 310 315 320 Asn Arg Ser Leu Ala Pro Thr Asn Leu Phe Ala Glu Lys Lys Gly Phe 325 330 335 Asn Phe Gly Phe Ala Thr Thr Tyr Ala Gln Leu 340 345 <210> 13 <211> 313 <212> PRT <213> Ricinus communis <400> 13 Met Ser Ser Ser Cys Ala Thr Val Thr Asn Val Tyr Glu Asn Gly Lys 1 5 10 15 Tyr Thr Thr Val Val Ala Lys Ile Glu Ser Gly Ser Cys Ala Arg Ser 20 25 30 Ser Leu Pro Leu Pro Leu Pro Pro Lys Pro Leu Leu Ile Ala Met Pro 35 40 45 Ser Glu Ala Gly Glu Phe Pro Val Leu Ile Phe Leu His Gly Tyr Leu 50 55 60 Leu Tyr Asn Ser Phe Tyr Ser Leu Leu Ile Gln His Val Ala Ser His 65 70 75 80 Gly Phe Ile Val Ile Ala Pro Gln Leu Tyr Thr Val Ala Gly Ala Asp 85 90 95 Ser Ala Asp Glu Ile Lys Cys Thr Ala Ala Ile Thr Asn Trp Leu Ser 100 105 110 Lys Gly Leu His His Val Leu Pro Pro His Val Gln Pro Lys Leu Ser 115 120 125 Lys Leu Gly Leu Ala Gly His Ser Arg Gly Gly Lys Ala Ala Phe Ala 130 135 140 Leu Ala Leu Gln Lys Ala Gly Ile Ser Thr Ala Leu Lys Phe Ser Ala 145 150 155 160 Leu Ile Gly Val Asp Pro Val Asp Gly Met Asp Lys Gly Lys Gln Thr 165 170 175 Pro Pro Pro Val Leu Thr Tyr Thr Pro His Ser Phe Asp Leu Asp Met 180 185 190 Ala Ala Met Val Ile Gly Ser Gly Leu Gly Glu Val Lys Arg Asn Pro 195 200 205 Met Phe Pro Pro Cys Ala Pro Lys Gly Val Asn His Glu Asp Phe Phe 210 215 220 Lys Glu Cys Lys Lys Pro Ala Tyr Tyr Phe Val Val Lys Asp Tyr Gly 225 230 235 240 His Leu Asp Met Leu Asp Asp Asp Thr Asn Gly Ile Arg Gly Lys Ala 245 250 255 Thr Tyr Cys Leu Cys Val Asn Gly Lys Ser Arg Glu Pro Met Arg Arg 260 265 270 Phe Val Gly Gly Val Leu Val Ala Phe Leu Lys Ala Tyr Leu Gly Gly 275 280 285 Asp Ser Ser Asp Leu Met Thr Ile Thr Asp Gly Gln Thr Gly Pro Val 290 295 300 Glu Leu Gln Ala Ala Glu Cys Tyr Val 305 310 <210> 14 <211> 316 <212> PRT <213> Glycine max <400> 14 Met Ala Gln Arg Ala Gln Pro Ala Leu Ala Thr Thr Asp Val Phe Gln 1 5 10 15 Lys Gly Asp Ile His Trp Lys Gln Phe Asn Val Glu Thr Ser Thr Ala 20 25 30 Ser Ser Ser Pro Pro Lys Pro Leu Leu Ile Phe Thr Pro Thr Val Pro 35 40 45 Gly Leu Tyr Pro Val Ile Leu Phe Cys His Gly Phe Cys Ile Arg Thr 50 55 60 Ser Tyr Tyr Ser Lys Leu Leu Ala His Ile Val Ser His Gly Phe Ile 65 70 75 80 Leu Val Ala Pro Gln Leu Phe Ser Ile Gly Val Pro Met Phe Gly Pro 85 90 95 Glu Glu Val Lys Cys Glu Gly Arg Val Val Asp Trp Leu Asp Asn Gly 100 105 110 Leu Gln Pro Leu Leu Pro Glu Ser Val Glu Ala Lys Leu Glu Lys Leu 115 120 125 Val Leu Val Gly His Ser Lys Gly Gly Lys Thr Ala Phe Ala Val Ala 130 135 140 Leu Gly Tyr Cys Lys Thr Lys Leu Lys Phe Ser Ala Leu Ile Gly Ile 145 150 155 160 Asp Pro Val Ala Gly Val Ser Lys Cys Lys Pro Cys Arg Ser Leu Pro 165 170 175 Asp Ile Leu Thr Gly Val Pro Arg Ser Phe Asn Leu Asn Ile Pro Val 180 185 190 Ala Val Ile Gly Thr Gly Leu Gly Pro Glu Lys Ala Asn Ser Leu Phe 195 200 205 Pro Pro Cys Ala Pro Asn Gly Val Asn His Lys Glu Phe Phe Ser Glu 210 215 220 Cys Lys Pro Pro Ser Ala Tyr Phe Val Ala Thr Asp Tyr Gly His Met 225 230 235 240 Asp Met Leu Asp Asp Glu Thr Pro Gly Val Ile Gly Thr Met Met Ser 245 250 255 Lys Cys Met Cys Lys Asn Gly Lys Lys Gly Pro Arg Asp Leu Met Arg 260 265 270 Arg Thr Val Gly Gly Leu Val Val Ala Phe Leu Arg Ala Gln Leu Asn 275 280 285 Glu Gln Trp Lys Asp Phe Asp Ala Ile Leu Ala Ser Pro Asn Leu Ala 290 295 300 Pro Ala Lys Leu Asp Asp Val Arg Tyr Leu Pro Thr 305 310 315 <210> 15 <211> 342 <212> PRT <213> Ginkgo biloba <400> 15 Met Val Leu Val Lys Asp Val Phe Ser Glu Gly Pro Leu Pro Val Gln 1 5 10 15 Ile Leu Ala Ile Pro Gln Ala Asn Ser Ser Pro Cys Ser Lys Leu Ala 20 25 30 Asp Lys Asn Gly Thr Ala Thr Thr Pro Ser Pro Cys Arg Pro Pro Lys 35 40 45 Pro Leu Leu Ile Ala Leu Pro Ser Gln His Gly Asp Tyr Pro Leu Ile 50 55 60 Leu Phe Phe His Gly Tyr Val Leu Leu Asn Ser Phe Tyr Ser Gln Leu 65 70 75 80 Leo Arg His Val Ala Ser His Gly Tyr Ile Ala Ile Ala Pro Gln Met 85 90 95 Tyr Ser Val Ile Gly Pro Asn Thr Thr Pro Glu Ile Ala Asp Ala Ala 100 105 110 Ala Ile Thr Asp Trp Leu Arg Asp Gly Leu Ser Asp Asn Leu Pro Gln 115 120 125 Ala Leu Asn Asn His Val Arg Pro Asn Phe Glu Lys Phe Val Leu Ala 130 135 140 Gly His Ser Arg Gly Gly Lys Val Ala Phe Ala Leu Ala Leu Gly Arg 145 150 155 160 Val Ser Gln Pro Ser Leu Lys Tyr Ser Ala Leu Val Gly Leu Asp Pro 165 170 175 Val Asp Gly Met Gly Lys Asp Gln Gln Thr Ser His Pro Ile Leu Ser 180 185 190 Tyr Arg Glu His Ser Phe Asp Leu Gly Met Pro Thr Leu Val Val Gly 195 200 205 Ser Gly Leu Gly Pro Cys Lys Arg Asn Pro Leu Phe Pro Pro Cys Ala 210 215 220 Pro Gln Gly Val Asn His His Asp Phe Phe Tyr Glu Cys Val Ala Pro 225 230 235 240 Ala Tyr His Phe Val Ala Ser Asp Tyr Gly His Leu Asp Phe Leu Asp 245 250 255 Asp Asp Thr Lys Gly Ile Arg Gly Lys Ala Thr Tyr Cys Leu Cys Lys 260 265 270 Asn Gly Glu Ala Arg Glu Pro Met Arg Lys Phe Ser Gly Gly Ile Val 275 280 285 Val Ala Phe Leu Gln Ala Phe Leu Gly Asp Asn Arg Gly Ala Leu Asn 290 295 300 Asp Ile Met Val Tyr Pro Ser His Ala Pro Val Lys Ile Glu Pro Pro 305 310 315 320 Glu Ser Leu Val Thr Glu Asp Val Lys Ser Pro Glu Val Glu Leu Leu 325 330 335 Arg Arg Ala Val Cys Arg 340 <210> 16 <211> 313 <212> PRT <213> Pachira macrocarpa <400> 16 Met Ala Gln Leu Leu Glu Thr Lys His Asp Leu Ser Thr Val Val Pro 1 5 10 15 Val Phe Val Thr Gly Lys Tyr His Pro Thr Ser Val Ser Val Asp Pro 20 25 30 Ser Asn Ser Ser Pro Ser Ser Pro Pro Lys Pro Leu Leu Ile Phe Thr 35 40 45 Pro Ser Glu Gln Gly Thr Tyr Pro Val Ile Leu Phe Phe His Gly Phe 50 55 60 Tyr Leu Arg Asn Asn Phe Tyr Thr Gly Leu Leu Leu His Ile Ser Ser 65 70 75 80 His Gly Phe Ile Ile Val Ala Pro Gln Leu Ser Asn Ile Ile Pro Pro 85 90 95 Ser Gly Thr Glu Glu Val Glu His Ala Ala Lys Val Ala Asp Trp Leu 100 105 110 Pro Ser Gly Leu Pro Ser Val Leu Pro Gly Asn Val Glu Ala Asn Leu 115 120 125 Ala Lys Leu Ala Leu Val Gly His Ser Arg Gly Gly Lys Thr Ala Phe 130 135 140 Ala Leu Ala Leu Gly Arg Ala Lys Thr Ala Gln Asn Phe Ser Ala Leu 145 150 155 160 Val Gly Ile Asp Pro Val Ala Gly Asn Arg Phe Gly Glu Thr Ser Pro 165 170 175 Lys Ile Leu Thr Tyr Thr Pro Gly Ser Phe Asp Leu Ser Ile Pro Val 180 185 190 Ala Val Val Gly Thr Gly Leu Gly Pro Glu Ser Lys Gly Cys Met Pro 195 200 205 Cys Pro Cys Ala Pro Thr Gln Tyr Asn His Glu Glu Phe Phe Asn Glu 210 215 220 Cys Lys Pro Pro Arg Val His Phe Asp Ala Lys Asn Tyr Gly His Met 225 230 235 240 Asp Thr Leu Asp Asp Asn Pro Ser Gly Phe Ile Gly Lys Leu Ser Asp 245 250 255 Thr Ile Cys Val Asn Gly Glu Gly Pro Arg Asp Pro Met Arg Arg Cys 260 265 270 Val Gly Gly Ile Val Val Ala Phe Leu Asn Tyr Phe Phe Glu Ala Glu 275 280 285 Lys Glu Asp Phe Met Thr Ile Met Asn Glu Pro Tyr Val Ala Pro Val 290 295 300 Thr Leu Asp Gln Val Gln Phe Asn Val 305 310 <210> 17 <211> 318 <212> PRT <213> Populus trichocarpa <400> 17 Met Ser Ser Ser Ser Ala Ile Ala Thr Val Thr Thr Thr Val Phe Glu 1 5 10 15 Ala Gly Lys Tyr Thr Thr Val Leu Gln Lys Val Glu Ser Arg Thr Thr 20 25 30 Cys Cys Thr Ala Lys Thr Ser Pro Pro Leu Pro Val Pro Pro Pro Lys 35 40 45 Pro Leu Leu Ile Val Met Pro Cys Glu Ala Gly Glu Phe Pro Leu Leu 50 55 60 Val Phe Leu His Gly Tyr Leu Leu Tyr Asn Ser Phe Tyr Ser Gln Leu 65 70 75 80 Leu Gln His Ile Ala Ser His Gly Phe Ile Val Ile Ala Pro Gln Leu 85 90 95 Tyr Leu Val Ala Gly Gln Asp Ser Ser Asp Glu Ile Lys Ser Val Ala 100 105 110 Ala Thr Thr Asn Trp Leu Ser Glu Gly Leu His His Leu Leu Pro Pro 115 120 125 His Val Lys Pro Asn Leu Ser Lys Leu Gly Leu Ala Gly His Ser Arg 130 135 140 Gly Gly Lys Thr Ala Phe Ala Leu Ala Leu Glu Lys Ala Ala Ala Thr 145 150 155 160 Leu Lys Phe Ser Ala Leu Ile Gly Val Asp Pro Val Asp Gly Met Asp 165 170 175 Lys Gly Lys Gln Thr Pro Pro Pro Val Leu Thr Tyr Val Pro His Ser 180 185 190 Phe Asp Leu Asp Met Ala Ile Met Val Ile Gly Ser Gly Leu Gly Glu 195 200 205 Leu Lys Lys Asn Pro Leu Phe Pro Pro Cys Ala Pro Glu Gly Val Asn 210 215 220 His Lys Asp Phe Phe Lys Glu Cys Lys Gly Pro Ala Ser Tyr Phe Val 225 230 235 240 Val Lys Asp Tyr Gly His Leu Asp Met Leu Asp Asp Asp Thr Glu Gly 245 250 255 Ile Arg Gly Lys Thr Thr Tyr Cys Leu Cys Lys Asn Gly Lys Ser Arg 260 265 270 Glu Pro Met Arg Lys Phe Ile Gly Gly Val Val Val Ala Phe Met Lys 275 280 285 Ala Tyr Leu Gly Gly Asp Ser Ser Asp Leu Met Ala Ile Lys Gly Gly 290 295 300 Gln Thr Gly Pro Val Glu Leu Gln Thr Val Glu Tyr Ile Leu 305 310 315 <210> 18 <211> 318 <212> PRT <213> Sorghum bicolor <400> 18 Met Ala Thr Thr Pro Lys Val Leu Glu Glu Pro Pro Ser Ala Val Ile 1 5 10 15 Thr Ser Val Phe Gln Pro Gly Lys Leu Ala Val Glu Val Ile Ser Val 20 25 30 Glu His Asp Ala Arg Pro Thr Pro Pro Pro Ile Pro Ile Leu Ile Ala 35 40 45 Ala Pro Lys Asp Ala Gly Thr Tyr Pro Val Ala Ile Leu Leu His Gly 50 55 60 Phe Phe Leu Gln Asn Arg Tyr Tyr Glu Gln Leu Leu Lys His Val Ala 65 70 75 80 Ser Phe Gly Phe Ile Met Val Ala Pro Gln Phe His Thr Ser Leu Ile 85 90 95 Ser Asn Ser Asp Ala Asp Asp Ile Ala Ala Ala Ala Lys Val Thr Asp 100 105 110 Trp Leu Pro Glu Gly Leu Pro Thr Val Leu Pro Thr Gly Val Glu Ala 115 120 125 Asp Leu Ser Lys Leu Ala Leu Ala Gly His Ser Arg Gly Gly His Thr 130 135 140 Ala Phe Ser Leu Ala Leu Gly Tyr Ala Lys Thr Asn Thr Ser Ser Leu 145 150 155 160 Leu Lys Phe Ser Ala Leu Ile Gly Leu Asp Pro Val Ala Gly Thr Gly 165 170 175 Lys Asn Ser Gln Leu Pro Pro Ala Ile Leu Thr Tyr Glu Pro Ser Ser 180 185 190 Phe Asp Ile Ala Val Pro Val Leu Val Ile Gly Thr Gly Leu Gly Asp 195 200 205 Glu Arg Glu Asn Ala Leu Phe Pro Pro Cys Ala Pro Val Glu Val Asn 210 215 220 His Ala Glu Phe Tyr Arg Glu Cys Arg Ala Pro Cys Tyr His Leu Val 225 230 235 240 Thr Lys Asp Tyr Gly His Leu Asp Met Leu Asp Asp Asp Ala Pro Lys 245 250 255 Leu Val Thr Cys Leu Cys Lys Glu Gly Asn Thr Cys Lys Asp Val Met 260 265 270 Arg Arg Thr Val Ala Gly Ile Met Val Ala Phe Leu Lys Ala Val Met 275 280 285 Gly Glu Asp Glu Asp Gly Asp Leu Lys Ala Ile Leu Gln His Pro Gly 290 295 300 Leu Ala Pro Thr Ile Leu Asp Pro Val Glu Tyr Arg Leu Ala 305 310 315 <210> 19 <211> 338 <212> PRT <213> Sorghum <400> 19 Met Ala Ser Pro Val Ala Ile Ser Thr Thr Ala Val Phe Lys Arg Gly 1 5 10 15 Arg His Pro Val Asp Thr Lys His Val Asp His Ser Gln Val Pro Gly 20 25 30 Val Pro Lys Pro Leu Met Val Val Thr Pro Thr Asp Ala Gly Val Tyr 35 40 45 Pro Val Ala Val Phe Leu His Gly Cys Ser Met Tyr Asn Ser Trp Tyr 50 55 60 Gln Thr Leu Leu Ser His Val Ala Ser His Gly Phe Ile Ala Val Ala 65 70 75 80 Pro Gln Leu Gly Gly Ile Leu Pro Pro Leu Asp Met Lys Asp Leu Lys 85 90 95 Asp Ile Asp Ala Thr Arg Lys Val Thr Ala Trp Leu Ala Asp Asn Leu 100 105 110 Ala His Val Leu Thr Asn Ile Leu His Leu His Gly Val Thr Pro Asp 115 120 125 Leu Ser Arg Leu Ala Leu Ala Gly His Ser Arg Gly Gly Asp Thr Ala 130 135 140 Phe Ala Val Ala Leu Gly Leu Gly Ser Ser Ser Ser Ser Ser Asp Thr 145 150 155 160 Thr Pro Leu Lys Phe Ser Ala Leu Ile Gly Val Asp Pro Val Ala Gly 165 170 175 Leu Ser Lys Glu Leu Gln Leu Glu Pro Lys Val Leu Thr Phe Glu Pro 180 185 190 Arg Ser Leu Asp Pro Gly Met Pro Ala Leu Val Val Gly Thr Gly Leu 195 200 205 Gly Pro Lys Gly Leu Leu Pro Cys Ala Pro Ala Gly Val Ser His Gly 210 215 220 Glu Phe Tyr Asp Glu Cys Ala Pro Pro Arg Tyr His Val Val Val Arg 225 230 235 240 Asp Tyr Gly His Leu Asp Met Leu Asp Asp Asp Gly Val Pro Tyr Val 245 250 255 Ile Ser Asn Cys Met Cys Lys Arg Asn Thr Asn Thr Thr Lys Asp Leu 260 265 270 Ala Arg Arg Ala Ile Gly Gly Ala Met Val Ala Phe Leu Arg Ala Lys 275 280 285 Leu Glu Asp Asp Asp Glu Asp Leu Arg Ala Val Leu Gln Asn Ser Pro 290 295 300 Gly Leu Ser Pro Ala Val Leu Asp Pro Val Glu Tyr Asp Asp Asp Glu 305 310 315 320 Ala Met Asp Gly Pro Gly Cys Ala Gly Asn Asn Gly Val Ala Gly Ala 325 330 335 Ser Gly <210> 20 <211> 319 <212> PRT <213> Vitis vinifera <400> 20 Met Ala Leu Leu Gly Gly Asn Pro Ser Thr Gln Gly Ile Lys Leu Asp 1 5 10 15 Leu Lys Thr Thr Thr Ser Val Phe Glu Pro Gly Asn Leu Ser Val Thr 20 25 30 Cys Ile Arg Val Glu Thr Ser Asn Ile Ala Ser Pro Pro Lys Pro Leu 35 40 45 Leu Ile Val Thr Pro Thr Ile Gln Gly Thr Tyr Pro Val Leu Leu Phe 50 55 60 Leu His Gly Phe Glu Leu Arg Asn Thr Phe Tyr Thr Gln Leu Leu Gln 65 70 75 80 Leu Ile Ser Ser His Gly Tyr Ile Val Val Ala Pro Gln Leu Tyr Gly 85 90 95 Leu Leu Pro Pro Ser Gly Ile Gln Glu Ile Lys Ser Ala Ala Ala Val 100 105 110 Thr Asn Trp Leu Ser Ser Gly Leu Gln Ser Val Leu Pro Glu Asn Val 115 120 125 Lys Pro Asp Leu Leu Lys Leu Ala Leu Ser Gly His Ser Arg Gly Gly 130 135 140 Lys Thr Ala Phe Ala Leu Ala Leu Gly Tyr Ala Asp Thr Ser Leu Asn 145 150 155 160 Phe Ser Ala Leu Leu Gly Leu Asp Pro Val Gly Gly Leu Ser Lys Cys 165 170 175 Ser Gln Thr Val Pro Lys Ile Leu Thr Tyr Val Pro His Ser Phe Asn 180 185 190 Leu Ala Ile Pro Val Cys Val Ile Gly Thr Gly Leu Gly Asp Glu Pro 195 200 205 Arg Asn Cys Leu Thr Cys Pro Cys Ala Pro Asp Gly Val Asn His Val 210 215 220 Glu Phe Phe Ser Glu Cys Lys Pro Pro Cys Ser His Phe Val Thr Thr 225 230 235 240 Glu Tyr Gly His Leu Asp Met Leu Asp Asp His Leu Ser Gly Cys Ile 245 250 255 Gly Ala Ile Ser Gly Tyr Ile Cys Lys Ser Gly Lys Gly Pro Arg Asp 260 265 270 Pro Met Arg Arg Cys Val Gly Gly Leu Phe Val Ala Phe Leu Lys Ala 275 280 285 Tyr Leu Glu Gly Gln Thr Gly Asp Phe Lys Ala Ile Val Asp Glu Pro 290 295 300 Asp Leu Ala Pro Val Lys Leu Asp Pro Val Glu Phe Ile Glu Ala 305 310 315 <210> 21 <211> 341 <212> PRT <213> Physcomitrella patens <400> 21 Met Glu Asp Pro Ile Pro Asn Val His Gly Gly Ile Tyr Glu Asp Gly 1 5 10 15 Pro Phe Lys Ile Glu Ile Val His Val Asp Asp Ala Ser Ser Ser Ser 20 25 30 Thr Cys Leu Lys Lys Ser Arg Ala Ala Val Asp Arg Glu Asn Leu Ser 35 40 45 Pro Lys Pro Leu Val Val Ala Leu Pro Lys Glu Glu Gly Val Tyr Pro 50 55 60 Val Ile Gln Phe His His Gly Phe Thr Leu Gln Asn Met Phe Tyr Ser 65 70 75 80 Gln Ile Ile Ser His Ile Ala Ser Tyr Gly Phe Ile Val Val Ala Pro 85 90 95 Gln Met Tyr Lys Ile Ser Gly Ser Asp Ala Thr Thr Glu Ile Glu Asp 100 105 110 Ala Val Gln Ile Leu Asn Trp Met Pro Thr Gly Leu Val Ala Ala Leu 115 120 125 Pro Glu Thr Leu Ser Lys His Arg Pro Asp Phe Ser Lys Val Ala Leu 130 135 140 Val Gly His Ser Arg Gly Ala Lys Val Val Phe Gly Leu Ala Leu Gly 145 150 155 160 Val Arg Asn Ser Ile Leu Gln Tyr Ser Ala Val Val Gly Leu Asp Pro 165 170 175 Val Asp Gly Met Gly Ile Gly Gln Gln Thr Asn Pro Pro Ile Leu Gln 180 185 190 Phe Ser Glu Gly Ser Leu Asn Leu Gly Val Pro Thr Leu Ile Ile Gly 195 200 205 Thr Gly Leu Gly Pro Leu Arg Lys Asn Phe Leu Phe Pro Ala Cys Ala 210 215 220 Pro Ala Gly Val Ser His Glu Ala Phe Tyr Tyr Asp Ser Ala Ala Pro 225 230 235 240 Ala Phe His Phe Val Ala Ser Lys Gln Gly His Met Asp Phe Leu Asn 245 250 255 Asp Asp Cys Ser Gly Pro Thr Gly Met Phe Ser Tyr Cys Leu Cys Lys 260 265 270 Asn Gly Pro Thr Arg Lys Pro Met Arg Arg Phe Ser Gly Gly Met Val 275 280 285 Val Ala Phe Leu Arg Ala Ala Phe Phe Gly Glu Thr Ala Pro Leu Val 290 295 300 Ala Ala Leu Ala Thr Pro Glu Leu Ala Pro Ile Pro Leu Asp Arg Pro 305 310 315 320 Glu Phe Lys Gly Lys Leu Gly Asp Ala Phe Asn Lys Pro Met Leu Ala 325 330 335 Pro Ala Leu Thr Pro 340 <210> 22 <211> 280 <212> PRT <213> Aquilegia sp. <400> 22 Met Thr Thr Ser Leu Pro Pro Pro Lys Pro Leu Leu Ile Ala Thr Pro 1 5 10 15 Ser Glu Glu Gly Gln Phe Pro Val Leu Ile Phe Leu His Gly Phe Leu 20 25 30 Leu Phe Asn Lys Phe Tyr Ser Gln Leu Ile Gln His Ile Ala Ser His 35 40 45 Gly Phe Ile Val Ile Ala Pro Gln Leu Tyr Lys Val Ala Gly Pro Asp 50 55 60 Thr Thr Asp Glu Ile Lys Ser Ala Ala Leu Val Ile Asp Trp Leu Ser 65 70 75 80 Asn Gly Leu His Ser Val Leu Pro Pro Leu Val Gln Pro Asn Leu Ser 85 90 95 Lys Leu Gly Ile Gly Gly His Ser Arg Gly Gly Lys Val Ala Phe Ala 100 105 110 Leu Ala Leu Gly His Ile Lys Thr Ser Leu Lys Tyr Ser Val Leu Leu 115 120 125 Gly Ile Asp Pro Val Asp Gly Met Gly Gln Gly Asn Gln Thr Pro Pro 130 135 140 Pro Val Leu Thr Tyr Thr Pro Arg Ser Phe Asp Phe Asn Met Pro Val 145 150 155 160 Leu Val Ile Gly Ser Gly Leu Gly Glu Thr Lys Lys Asn Ser Leu Phe 165 170 175 Pro Pro Cys Ala Pro Lys Gly Val Asn His Glu Asn Phe Tyr Ser Glu 180 185 190 Cys Cys Ser Pro Ala Cys Tyr Phe Val Val Lys Asp Tyr Gly His Met 195 200 205 Asp Met Leu Asp Asp Asp Thr Gly Gly Val Arg Gly Lys Ala Thr Tyr 210 215 220 Cys Thr Cys Ser Asn Gly Lys Ala Arg Glu Pro Met Arg Thr Phe Val 225 230 235 240 Gly Gly Ile Met Val Ala Phe Met Lys Ala Tyr Met Glu Asn Asp Ser 245 250 255 Arg Asp Leu Met Ala Ile Lys Glu Thr Gln Gly Met Ala Leu Ile Glu 260 265 270 Leu Gln Ser Val Glu Phe Arg Leu 275 280 <210> 23 <211> 324 <212> PRT <213> Brachypodium distachyon <400> 23 Met Ala Ala Thr Ala Ala Ala Ala Glu Leu Lys Lys Asn Ser Gly Ala 1 5 10 15 Asp Val Leu Glu Ala Val Ile Thr Ser Val Phe Gln Pro Gly Lys Leu 20 25 30 Ala Val Glu Val Ile Gln Val Asp His Asn Ala Val Pro Thr Pro Pro 35 40 45 Ile Pro Val Leu Ile Val Ala Pro Lys Asp Ala Gly Thr Tyr Pro Val 50 55 60 Ala Met Leu Leu His Gly Phe Phe Leu Gln Asn His Tyr Tyr Lys Gln 65 70 75 80 Leu Leu Arg His Val Ala Ser His Gly Phe Ile Met Val Ala Pro Gln 85 90 95 Phe His Leu Ser Met Ile Pro Thr Gly Asp Thr Lys Asp Ile Glu Ala 100 105 110 Ala Ala Lys Val Ser Asp Trp Leu Pro Glu Gly Leu Pro Ser Val Leu 115 120 125 Pro Lys Gly Val Glu Pro Glu Leu Ser Lys Leu Ala Leu Ala Gly His 130 135 140 Ser Arg Gly Gly His Thr Ala Phe Ser Leu Ala Leu Gly His Ala Lys 145 150 155 160 Ser Asn Leu Ser Phe Ser Ala Leu Ile Gly Ile Asp Pro Val Ala Gly 165 170 175 Thr Gly Lys Ser Ser Gln Leu Ala Pro Lys Ile Leu Thr Tyr Glu Pro 180 185 190 Ser Ser Phe Asn Met Ser Ala Ala Met Pro Val Leu Val Ile Gly Thr 195 200 205 Gly Leu Gly Glu Glu Lys Lys Asn Ile Phe Thr Pro Pro Cys Ala Pro 210 215 220 Lys Asp Val Asn His Arg Glu Phe Tyr Leu Glu Cys Lys Pro Pro Cys 225 230 235 240 Tyr Tyr Phe Val Thr Lys Asp Tyr Gly His Leu Asp Met Leu Asp Asp 245 250 255 Asp Ala Pro Met Val Ile Thr Cys Leu Cys Lys Asp Gly Gly Ser Cys 260 265 270 Lys Asp Lys Met Arg Arg Cys Val Ala Gly Ile Met Val Ala Phe Leu 275 280 285 Asn Ser Ala Leu Gly Gly Lys Asp Asn Ala Ala His Asp Leu Glu Val 290 295 300 Ile Val Lys Asp Pro Ala Leu Ala Pro Thr Thr Leu Asp Pro Val Glu 305 310 315 320 Cys Arg Leu Glu <210> 24 <211> 306 <212> PRT <213> Medicago truncatula <400> 24 Met Cys Ser Ser Val Ser Asn Val Phe Glu Thr Gly Asn Tyr Thr Thr 1 5 10 15 Lys Leu Leu Arg Val Asp Ser Cys Ser His Ala Gln Asn Val Pro Pro 20 25 30 Pro Lys Ser Leu Leu Ile Ala Thr Pro Ile Glu Gly Gly Glu Phe Pro 35 40 45 Leu Leu Leu Phe Leu His Gly Tyr Leu Leu Leu Asn Ser Phe Tyr Ser 50 55 60 Gln Leu Ile Gln His Val Ala Ser His Gly Phe Ile Val Ile Ala Pro 65 70 75 80 Gln Leu Tyr Thr Val Ala Gly Pro Asp Ile Thr Glu Glu Ile Tyr Ser 85 90 95 Val Ala Ala Ile Thr Asn Trp Leu Ser Lys Gly Leu Ser Lys Ile Leu 100 105 110 Pro Leu Asn Ile Lys Pro Asn Phe His Lys Leu Ala Leu Gly Gly His 115 120 125 Ser Arg Gly Gly Lys Thr Ser Phe Ala Val Ala Leu Arg Lys Leu Asn 130 135 140 Met Thr Thr Asp Leu Lys Phe Ser Ala Ile Ile Gly Val Asp Pro Val 145 150 155 160 Asp Gly Met Asp Lys Gly Lys Gln Thr Ser Pro Pro Ile Phe Thr Tyr 165 170 175 Val Pro His Ser Phe Asp Tyr Asp Met Ala Thr Leu Val Ile Gly Phe 180 185 190 Gly Leu Gly Asp Val Lys Lys Asn Pro Leu Phe Pro Pro Cys Ala Pro 195 200 205 Lys Gly Val Asn His Glu Asp Phe Phe Ser Glu Cys Glu Lys Pro Ser 210 215 220 Trp Tyr Phe Val Ala Lys Asp Tyr Gly His Val Asp Met Leu Asp Asp 225 230 235 240 Asp Thr Lys Gly Val Arg Gly Lys Val Ser Tyr Cys Leu Cys Lys Asn 245 250 255 Gly Glu Ser Arg Lys Pro Met Arg Met Phe Val Gly Gly Val Met Val 260 265 270 Ala Phe Leu Lys Ala Tyr Leu His Gly Asp Asn Val Asp Leu Leu Ala 275 280 285 Ile Arg Asp Lys Asn Leu Ser Val Pro Ile Glu Met Lys Phe Asp Tyr 290 295 300 Phe Val 305 <210> 25 <211> 306 <212> PRT <213> Piper betle <400> 25 Met Ala Ala Ser Ser Val Phe Glu Met Gly Lys Leu Glu Val His Val 1 5 10 15 Lys Ser Val Asn Gln Ser Asn Ser Ser Ser Pro Pro Lys Ser Leu Leu 20 25 30 Ile Ser Tyr Pro Ser Gln Lys Gly Asp Tyr Gly Val Val Leu Phe Leu 35 40 45 His Gly Phe Leu Ile Ser Asn Ser Phe Tyr Lys Glu Leu Ile Ser His 50 55 60 Ile Ser Ser His Gly Tyr Ile Val Val Ala Pro Arg Ile Ile Tyr Pro 65 70 75 80 Cys Leu Gln Asp Glu Ile Asn Ser Ala Ala Gln Val Ala Asn Trp Leu 85 90 95 Pro Glu Gly Leu Gln Ala Ala Leu Pro Pro Asn Val Gln Pro Asn Thr 100 105 110 Ser Lys Leu Thr Leu Ala Gly His Ser Arg Gly Gly Lys Ala Ala Phe 115 120 125 Cys Met Leu Leu Gly Leu Ala Gly Ser Pro Leu Thr Val Gln Phe Ser 130 135 140 Gly Leu Ile Gly Val Asp Pro Val Ala Gly Phe Gln Ile Pro Gly Ile 145 150 155 160 Asn Tyr Lys Met Glu Ile Pro Pro Lys Ile Ile Thr Asn Asn Ser Lys 165 170 175 Pro Phe Asp Ile Asn Val Pro Thr Leu Ile Ile Gly Thr Glu Leu Gly 180 185 190 Glu Glu Ala Lys Gly Cys Leu Ala Pro Pro Tyr Ala Pro Ala Gly Leu 195 200 205 Asn Tyr Glu Gln Phe Tyr Glu Lys Ser Lys Glu Pro Ser Tyr Gln Phe 210 215 220 Val Ala Lys Gly Tyr Gly His Val Asp Met Leu Asp Asp Ile Ser Lys 225 230 235 240 Asn Asp Leu Met Gly Lys Leu Thr Tyr Cys Val Cys Lys Asn Gly Lys 245 250 255 Glu Arg Glu Pro Met Arg Arg Thr Ala Gly Gly Leu Met Val Ala Phe 260 265 270 Leu Lys Ala Phe Ser Asp Gly Gln Arg Asp Asp Leu Asp Ala Ile Leu 275 280 285 Asn Asp Pro Glu Leu Ala Pro Ile Gln Leu Asp Ala Gly Ala Lys Leu 290 295 300 Ser Ser<000208​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Ile Val Ile Ala Pro Gln Leu Tyr Ala Val Ala Gly Pro Asp Val Ser 100 105 110 Gly Glu Ile His Ser Thr Ala Ala Ile Lys Asn Trp Leu Ser Glu Gly 115 120 125 Leu Ser Lys Phe Leu Pro Pro Asn Val Thr Pro Asn Ser Ser Lys Leu 130 135 140 Ala Leu Ala Gly His Ser Arg Gly Gly Lys Thr Ala Phe Ala Val Ala 145 150 155 160 Leu Arg Lys Leu Asn Ile Thr Thr Asp Leu Lys Phe Ser Ala Leu Val 165 170 175 Gly Val Asp Pro Val Asp Gly Leu Asp Arg Gly Lys Gln Thr Pro Pro 180 185 190 Pro Val Leu Thr Tyr Val Pro His Ser Phe Asp Phe Asp Met Pro Ala 195 200 205 Met Val Ile Gly Ser Gly Leu Gly Asp Val Lys Arg Asn Pro Leu Phe 210 215 220 Pro Pro Cys Ala Pro Lys Thr Val Asn His Glu Asp Phe Phe Asn Glu 225 230 235 240 Cys Asn Lys Pro Ala Trp Tyr Phe Val Ala Lys Asp Tyr Gly His Val 245 250 255 Asp Met Leu Asp Asp Asp Thr Asn Gly Ile Ile Gly Lys Ala Thr Tyr 260 265 270 Cys Leu Cys Lys Asn Gly Glu Ser Arg Lys Pro Met Arg Thr Phe Val 275 280 285 Gly Gly Leu Val Val Ala Phe Leu Lys Ala Tyr Leu Gln Gly Asp Asn 290 295 300 Arg Asp Ser Leu Ala Ile Lys Asp Lys His Leu Ser Ala Pro Val Glu 305 310 315 320 Leu Lys Phe Asp Tyr Phe Val 325 <210> 27 <211> 337 <212> PRT <213> Indica rice (Oryza sativa Indica) <400> 27 Met Ile Ala Phe Ala Ala Gln Ile Leu Ala Phe Cys Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Gln Leu Gln Thr Thr Met Ala Gly Asp Ser Ser 20 25 30 Phe Ser Gly Val Phe Asp His Gly Ser His Gly Val Thr Leu Val Lys 35 40 45 Val Asp Glu Ala Pro Arg Lys Cys Ser Ser Ala Ala Ala Ala Lys Lys 50 55 60 Thr Asp Asp Asp Thr Ala Pro Ala Gly Gly Ala Pro Pro Lys Pro Leu 65 70 75 80 Leu Val Ala Ala Pro Cys Asp Ala Gly Val Tyr Pro Val Val Val Phe 85 90 95 Leu His Gly Tyr Leu Ala Tyr Asn Ser Phe Tyr Ser Gln Leu Phe Glu 100 105 110 His Val Ala Ser His Gly Phe Val Val Val Gly Pro Gln Val Asn Gln 115 120 125 Ser Ile Leu Ile Tyr Tyr Phe Ser Tyr Ile Arg Cys Leu Asp Arg Ile 130 135 140 Pro Pro Thr Arg Ser Thr Arg Arg Ala Ala Val Ile Asn Trp Leu Ala 145 150 155 160 Ala Gly Gly Leu Thr Ser Lys Leu Pro Pro Asn Val Arg Ala Asp Ala 165 170 175 Thr Lys Ile Ser Ile Ser Gly His Ser Arg Gly Gly Lys Val Ala Phe 180 185 190 Ala Leu Ala Leu Gly His Ala Asn Val Ser Leu Arg Gly Gly Ala Gly 195 200 205 Gly Ala Thr Ile Ala Ala Leu Val Ala Val Asp Pro Val Asp Gly Phe 210 215 220 Ala Thr Gly Lys Gln Thr Pro Pro Pro Ile Leu Thr Tyr Gly Gly Ala 225 230 235 240 Asn Ser Leu Arg Val Pro Ala Pro Val Met Val Ile Gly Thr Gly Leu 245 250 255 Gly Gly Leu Ala Arg Ala Ala Pro Leu Leu Pro Ala Cys Ala Pro Pro 260 265 270 Gly Val Ser His Gly Glu Phe Tyr Gly Glu Cys Ala Ala Pro Ala Cys 275 280 285 His Leu Val Ala Arg Asp Tyr Gly His Thr Asp Met Val Val Asp Val 290 295 300 Thr Pro Gly Ser Trp Ala Ser Leu Arg Val Pro Cys Ala Gly Ala Ser 305 310 315 320 Ala Pro Gly Arg Pro Cys Val Gly Ser Ser Ser Ala Pro Trp Ser Arg 325 330 335 Ser <210> 28 <211> 367 <212> PRT <213> Japonica rice (Oryza sativa Japonica) <400> 28 Met Ile Ala Phe Ala Ala Gln Ile Leu Ala Phe Cys Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Gln Leu Gln Thr Thr Met Ala Gly Asp Ser Ser 20 25 30 Phe Ser Gly Val Phe Asp His Gly Ser His Gly Val Thr Leu Val Lys 35 40 45 Val Asp Glu Ala Pro Arg Lys Cys Ser Ser Ala Ala Ala Ala Lys Lys 50 55 60 Thr Asp Asp Asp Thr Ala Pro Ala Gly Gly Ala Pro Pro Lys Pro Leu 65 70 75 80 Leu Val Ala Ala Pro Cys Asp Ala Gly Val Tyr Pro Val Val Val Phe 85 90 95 Leu His Gly Tyr Leu Ala Tyr Asn Ser Phe Tyr Ser Gln Leu Phe Glu 100 105 110 His Val Ala Ser His Gly Phe Val Val Val Gly Pro Gln Leu Tyr Thr 115 120 125 Met Ser Gly Pro Asp Thr Thr Asp Glu Ile Asn Ser Ala Ala Ala Val 130 135 140 Ile Asn Trp Leu Ala Ala Gly Gly Leu Thr Ser Lys Leu Pro Pro Asn 145 150 155 160 Val Arg Ala Asp Ala Thr Lys Ile Ser Ile Ser Gly His Ser Arg Gly 165 170 175 Gly Lys Val Ala Phe Ala Leu Ala Leu Gly His Ala Asn Val Ser Leu 180 185 190 Arg Gly Gly Ala Gly Gly Ala Thr Ile Ala Ala Leu Val Ala Val Asp 195 200 205 Pro Val Asp Gly Phe Ala Ala Gly Lys Gln Thr Pro Pro Pro Ile Leu 210 215 220 Thr Tyr Gly Gly Ala Asn Ser Leu Arg Val Pro Ala Pro Val Met Val 225 230 235 240 Ile Gly Thr Gly Leu Gly Gly Leu Ala Arg Ala Ala Pro Leu Leu Pro 245 250 255 Ala Cys Ala Pro Pro Gly Val Ser His Gly Glu Phe Tyr Gly Glu Cys 260 265 270 Ala Ala Pro Ala Cys His Leu Val Ala Arg Asp Tyr Gly His Thr Asp 275 280 285 Met Met Asp Asp Val Thr Pro Gly Ala Arg Gly Leu Ala Thr Arg Ala 290 295 300 Val Cys Arg Ser Gly Gly Ala Arg Ala Pro Met Arg Arg Phe Phe Gly 305 310 315 320 Gly Ala Met Val Ala Phe Val Lys Arg Trp Val Glu Gly Glu Pro Glu 325 330 335 Leu Leu Asp Cys Val Arg Ala Arg Pro Glu Thr Ala Pro Val Val Leu 340 345 350 Ser Ala Val Glu Phe Arg Asp Glu Ala Ile Ala Asn His Ser Tyr 355 360 365 <210> 29 <211> 356 <212> PRT <213> Japonica rice <400> 29 Met Ile Ala Phe Ala Ala Gln Ile Leu Ala Phe Cys Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Gln Leu Gln Thr Thr Met Ala Gly Asp Ser Ser 20 25 30 Phe Ser Gly Val Phe Asp His Gly Ser His Gly Val Thr Leu Val Lys 35 40 45 Val Asp Glu Ala Pro Arg Lys Cys Ser Ser Ala Ala Ala Ala Lys Lys 50 55 60 Thr Asp Asp Asp Thr Ala Pro Ala Gly Gly Ala Pro Pro Lys Pro Leu 65 70 75 80 Leu Val Ala Ala Pro Cys Asp Ala Gly Val Tyr Pro Val Val Val Phe 85 90 95 Leu His Gly Tyr Leu Ala Tyr Asn Ser Phe Tyr Ser Gln Leu Phe Glu 100 105 110 His Val Ala Ser His Gly Phe Val Val Val Gly Pro Gln Leu Phe Leu 115 120 125 Gly Cys Glu Leu Ile Leu Ser Asn Asn Phe Asp Ala Lys Met Leu Tyr 130 135 140 Thr Met Ser Gly Pro Asp Thr Thr Asp Glu Ile Asn Ser Ala Ala Ala 145 150 155 160 Val Ile Asn Trp Leu Ala Ala Gly Gly Leu Thr Ser Lys Leu Pro Pro 165 170 175 Asn Val Arg Ala Asp Ala Thr Lys Ile Ser Ile Ser Gly His Ser Arg 180 185 190 Gly Gly Lys Val Ala Phe Ala Leu Ala Leu Gly His Ala Asn Gln Thr 195 200 205 Pro Arg Pro Ile Leu Thr Tyr Gly Gly Ala Asn Ser Leu Arg Leu Pro 210 215 220 Ala Pro Val Met Val Ile Gly Thr Gly Leu Gly Gly Leu Ala Arg Ala 225 230 235 240 Ala Pro Leu Leu Pro Ala Cys Ala Pro Pro Gly Val Ser His Gly Glu 245 250 255 Phe Tyr Gly Glu Cys Ala Ala Pro Ala Cys His Leu Val Ala Arg Asp 260 265 270 Tyr Gly His Thr Asp Met Met Asp Asp Val Thr Pro Gly Ala Arg Gly 275 280 285 Leu Ala Thr Arg Ala Val Cys Arg Ser Gly Gly Ala Arg Ala Pro Met 290 295 300 Arg Arg Phe Phe Gly Gly Ala Met Val Ala Phe Val Lys Arg Trp Val 305 310 315 320 Glu Gly Glu Pro Glu Leu Leu Asp Cys Val Arg Ala Arg Pro Glu Thr 325 330 335 Ala Pro Val Val Leu Ser Ala Val Glu Phe Arg Asp Glu Ala Ile Ala 340 345 350 Asn His Ser Tyr 355 <210> 30 <211> 329 <212> PRT <213> Picea sitchensis <400> 30 Met Gly Gln Gln Gly Glu Glu Pro Trp Glu Asp Val Phe Lys Pro Gly 1 5 10 15 Arg Phe Pro Val Arg Ile Leu Lys Ile Pro Gln Arg Thr Thr His Gly 20 25 30 Ser Thr Thr Ala Ala Ala Pro Lys Pro Leu Leu Leu Ala Leu Pro Ala 35 40 45 Gln Pro Gly Glu Tyr Pro Val Leu Leu Phe Phe His Gly Tyr Leu Leu 50 55 60 Leu Asn Ser Phe Tyr Thr Gln Leu Leu Gln His Ile Ala Ser His Gly 65 70 75 80 Tyr Ile Ala Ile Ala Pro Gln Met Tyr Cys Val Thr Gly Ala Asp Ala 85 90 95 Thr Pro Glu Ile Ala Asp Ala Ala Ala Ile Cys Asn Trp Leu Leu Gln 100 105 110 Gly Leu Ser Ser Tyr Leu Pro Asp Asp Val Arg Pro Asp Phe Gln Asn 115 120 125 Val Ala Met Ala Gly His Ser Arg Gly Gly Lys Val Ala Phe Gly Leu 130 135 140 Ala Leu Asp Arg Thr Ser Gln Thr Thr Glu Leu Lys Phe Ser Ala Leu 145 150 155 160 Val Gly Val Asp Pro Val Asp Gly Met Ala Arg Gly Arg Gln Thr Gln 165 170 175 Pro Arg Ile Leu Thr Tyr Lys Pro His Ser Phe Asp Ser Val Ile Pro 180 185 190 Thr Leu Ile Val Gly Ser Gly Leu Gly Ala Val Lys Arg Asn Pro Leu 195 200 205 Phe Pro Pro Cys Ala Pro Glu Gly Val Ser His Arg Glu Phe Phe Ser 210 215 220 Glu Cys Ser Ala Pro Ala Tyr His Phe Val Ala Ser Asp Tyr Gly His 225 230 235 240 Met Asp Phe Leu Asp Asp Glu Thr Gly Gly Val Lys Gly Gln Ser Ser 245 250 255 Tyr Cys Leu Cys Lys Asn Gly Val Ala Arg Glu Pro Met Arg Arg Phe 260 265 270 Cys Gly Gly Ile Ile Val Ala Phe Leu Asn Val Cys Leu Gln Asn Asp 275 280 285 Ser Gly Ala Phe Asn Asp Leu Leu Val His Pro Ser His Ala Pro Val 290 295 300 Lys Leu Glu Pro Pro Glu Ser Phe Val Ser Glu Val Glu His Gln Ala 305 310 315 320 Val Glu Ser Leu Leu Pro Gln Thr Val 325 <210> 31 <211> 322 <212> PRT <213> Chlamydomonas sp. <400> 31 Met Pro Ser Thr Gln Phe Leu Gly Ala Ser Thr Leu Leu Leu Phe Gly 1 5 10 15 Leu Arg Ala Val Met Ser Ser Asp Asp Tyr Ile Lys Arg Gly Asp Leu 20 25 30 Pro Thr Ser Lys Trp Ser Gly Arg Val Thr Leu Arg Val Asp Ser Ala 35 40 45 Met Ala Val Pro Leu Asp Val Val Ile Thr Tyr Pro Ser Ser Gly Ala 50 55 60 Ala Ala Tyr Pro Val Leu Val Met Tyr Asn Gly Phe Gln Ala Lys Ala 65 70 75 80 Pro Trp Tyr Arg Gly Ile Val Asp His Val Ser Ser Trp Gly Tyr Thr 85 90 95 Val Val Gln Tyr Thr Asn Gly Gly Leu Phe Pro Ile Val Val Asp Arg 100 105 110 Val Glu Leu Thr Tyr Leu Glu Pro Leu Leu Thr Trp Leu Glu Thr Gln 115 120 125 Ser Ala Asp Ala Lys Ser Pro Leu Tyr Gly Arg Ala Asp Val Ser Arg 130 135 140 Leu Gly Thr Met Gly His Ser Arg Gly Gly Lys Leu Ala Ala Leu Gln 145 150 155 160 Phe Ala Gly Arg Thr Asp Val Ser Gly Cys Val Leu Phe Asp Pro Val 165 170 175 Asp Gly Ser Pro Met Thr Pro Glu Ser Ala Asp Tyr Pro Ser Ala Thr 180 185 190 Lys Ala Leu Ala Ala Ala Gly Arg Ser Ala Gly Leu Val Gly Ala Ala 195 200 205 Ile Thr Gly Ser Cys Asn Pro Val Gly Gln Asn Tyr Pro Lys Phe Trp<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <220> <223> Conservative sequence motifs <400> 32 Gly His Ser Arg Gly 1 5 <210> 33 <211> 4 <212> PRT <213> Artificial sequence <220> <223> amino acid sequence motif <220> <221> variants <222> (4)..(4) <223> Xaa can be Leu, Ala, Val, Ile, Phe, Tyr, His, Gln, Thr, Asn, Met, or Ser. <400> 33 Gly Asp Ser Xaa 1 <210> 34 <211> 78 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequences showing the fusion of the chlorophyllase gene with histidine tags and thrombin sites. <400> 34 catgggcagc agccatcatc atcatcatca cagcagcggc ctggtgccgc gcggcagcca 60 tatggcagcg gctgcccc 78 <210> 35 <211> 26 <212> PRT <213> Artificial sequence <220> <223> The amino acid sequence showing the fusion of the chlorophyllase gene with the histidine tag and thrombin site. <400> 35 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ala Ala Ala Pro 20 25 <210> 36 <211> 155 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequences showing the fusion of the chlorophyllase gene with the AprE signal sequence and the AGK sequence. <400> 36 atttttttaa aaggagaggg taaagagtga gaagcaaaaa attgtggatc agtttgctgt 60 ttgctttagc gttaatcttt acgatggcgt tcggcagcac atccagcgcg caggctgctg 120 gaaaaatggc agcggctgcc ccggccgaaa caatg 155 <210> 37 <211> 43 <212> PRT <213> Artificial sequence <220> <223> The amino acid sequence showing the fusion of the chlorophyllase gene with the AprE signal sequence and the AGK sequence. <400> 37 Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Ala Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Gly Ser Thr Ser Ser Ala Gln Ala Ala Gly 20 25 30 Lys Met Ala Ala Ala Ala Pro Ala Glu Thr Met 35 40 <210> 38 <211> 77 <212> DNA <213> Artificial sequence <220> <223> The nucleotide sequence showing the direct fusion of the chlorophyllase gene with the AprE promoter. <400> 38 taagtaagtc tactctgaat ttttttaaaa ggagagggta actagtggca gcggctgccc 60 cggccgaaac aatgaat 77 <210> 39 <211> 11 <212> PRT <213> Artificial sequence <220> <223> The amino acid sequence showing the direct fusion of the chlorophyllase gene with the AprE promoter. <400> 39 Met Ala Ala Ala Ala Pro Ala Glu Thr Met Asn 1 5 10 <210> 40 <211> 156 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence showing the fusion of the chlorophyllase gene with the Cel A signal sequence. <400> 40 aaccatgggc tttggggagcg ctcccatcgc gttgtgtccg cttcgcacga ggaggacgc 60 tttgaaacgc cttttggccc tgctcgcgac cggcgtgtcg atcgtcggcc tgactgcgct 120 agccggcccc ccggcacagg ccatggccgc cgccgc 156 <210> 41 <211> 51 <212> PRT <213> Artificial sequence <220> <223> The amino acid sequence showing the fusion of the chlorophyllase gene with the Cel A signal sequence. <400> 41 Met Gly Phe Gly Ser Ala Pro Ile Ala Leu Cys Pro Leu Arg Thr Arg 1 5 10 15 Arg Asn Ala Leu Lys Arg Leu Leu Ala Leu Leu Ala Thr Gly Val Ser 20 25 30 Ile Val Gly Leu Thr Ala Leu Ala Gly Pro Pro Ala Gln Ala Met Ala 35 40 45 Ala Ala Ala 50

Claims

1. A method for processing vegetable oil, comprising the step of contacting the oil with an enzyme, wherein the enzyme is capable of hydrolyzing chlorophyll derivatives. a' A stereoisomer, wherein the chlorophyll derivative is pheophytin, and the enzyme is produced by using a nucleotide sequence encoding a polypeptide sequence of SEQ ID NO:

2.

2. The method of claim 1, wherein after treatment with the enzyme, the oil comprises based on the chlorophyll derivative in the oil. a and a' The total amount of stereoisomers, at least 50% of the chlorophyll derivative. a Stereoisomer.

3. A vegetable oil, which can be obtained by the method according to any one of the preceding claims, wherein the concentration of phytochlorophyll, pheophytin and / or pyrophyllin in the vegetable oil is less than 0.02 mg / kg.

4. Enzymes capable of hydrolyzing chlorophyll derivatives are used to remove pheophytin from vegetable oils. a' Use of stereoisomers, wherein the enzyme is produced by using a nucleotide sequence encoding a polypeptide sequence of SEQ ID NO:2.

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