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WO2012114232A1 - Procédé - Google Patents

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Publication number
WO2012114232A1
WO2012114232A1 PCT/IB2012/050712 IB2012050712W WO2012114232A1 WO 2012114232 A1 WO2012114232 A1 WO 2012114232A1 IB 2012050712 W IB2012050712 W IB 2012050712W WO 2012114232 A1 WO2012114232 A1 WO 2012114232A1
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WIPO (PCT)
Prior art keywords
oil
enzyme
chlorophyll
chlorophyllase
degumming
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/IB2012/050712
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English (en)
Inventor
Rene Mikkelsen
Jørn Borch Søe
Tina Lillan JØRGENSEN
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International N&H Denmark ApS
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DuPont Nutrition Biosciences ApS
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Publication of WO2012114232A1 publication Critical patent/WO2012114232A1/fr
Anticipated expiration legal-status Critical
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/003—Refining fats or fatty oils by enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/02—Refining fats or fatty oils by chemical reaction
    • C11B3/04—Refining fats or fatty oils by chemical reaction with acids
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/02—Refining fats or fatty oils by chemical reaction
    • C11B3/06—Refining fats or fatty oils by chemical reaction with bases
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/10—Refining fats or fatty oils by adsorption
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01014—Chlorophyllase (3.1.1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01082—Pheophorbidase (3.1.1.82)

Definitions

  • the present invention relates to the industrial processing of plant-derived food and feed products, especially vegetable oils.
  • the invention may be employed to reduce or eliminate contamination by chlorophyll and chlorophyll derivatives.
  • Chlorophyll is a green-coloured pigment widely found throughout the plant kingdom. Chlorophyll is essential for photosynthesis and is one of the most abundant organic metal compounds found on earth. Thus many products derived from plants, including foods and feeds, contain significant amounts of chlorophyll.
  • oils derived from oilseeds such as soybean, palm or rape seed (canola), cotton seed and peanut oil typically contain some chlorophyll.
  • chlorophyll pigments in vegetable oils is generally undesirable. This is because chlorophyll imparts an undesirable green colour and can induce oxidation of oil during storage, leading to a deterioration of the oil.
  • Chlorophyll may be removed during many stages of the oil production process, including the seed crushing, oil extraction, degumming, caustic treatment and bleaching steps.
  • the bleaching step is usually the most significant for reducing chlorophyll residues to an acceptable level.
  • the adsorbent used in the bleaching step is typically clay.
  • the use of such steps typically reduces chlorophyll levels in processed oil to between 0.02 to 0.05 ppm.
  • the bleaching step increases processing cost and reduces oil yield due to entrainment in the bleaching clay.
  • the use of clay may remove many desirable compounds such as carotenoids and tocopherol from the oil.
  • the use of clay is expensive, this is particularly due to the treatment of the used clay (i.e. the waste) which can be difficult, dangerous (prone to self-ignition) and thus costly to handle.
  • attempts have been made to remove chlorophyll from oil by other means, for instance using the enzyme chlorophyllase.
  • chlorophyllase In plants, chlorophyllase (chlase) is thought to be involved in chlorophyll degradation and catalyzes the hydrolysis of an ester bond in chlorophyll to yield chlorophyllide and phytol.
  • WO 2006009676 describes an industrial process in which chlorophyll contamination can be reduced in a composition such as a plant oil by treatment with chlorophyllase.
  • the water-soluble chlorophyllide which is produced in this process is also green in colour but can be removed by an aqueous extraction or silica treatment.
  • Chlorophyll is often partly degraded in the seeds used for oil production as well as during extraction of the oil from the seeds.
  • One common modification is the loss of the magnesium ion from the porphyrin (chlorin) ring to form the derivative known as pheophytin (see Figure 1).
  • the loss of the highly polar magnesium ion from the porphyrin ring results in significantly different physico-chemical properties of pheophytin compared to chlorophyll.
  • pheophytin is more abundant in the oil during processing than chlorophyll.
  • Pheophytin has a greenish colour and may be removed from the oil by an analogous process to that used for chlorophyll, for instance as described in WO 2006009676 by an esterase reaction catalyzed by an enzyme having a pheophytinase activity. Under certain conditions, some chlorophyllases are capable of hydrolyzing pheophytin as well as chlorophyll, and so are suitable for removing both of these contaminants. The products of pheophytin hydrolysis are the red/brown-colored pheophorbide and phytol. Pheophorbide can also be produced by the loss of a magnesium ion from chlorophyllide, i.e. following hydrolysis of chlorophyll (see Figure 1). WO 2006009676 teaches removal of pheophorbide by an analogous method to chlorophyllide, e.g. by aqueous extraction or silica adsorption.
  • Pheophytin may be further degraded to pyropheophytin, both by the activity of plant enzymes during harvest and storage of oil seeds or by processing conditions (e.g. heat) during oil refining (see “Behaviour of Chlorophyll Derivatives in Canola Oil Processing", JAOCS, Vol, no. 9 (Sept. 1993) pages 837-841).
  • processing conditions e.g. heat
  • One possible mechanism is the enzymatic hydrolysis of the methyl ester bond of the isocyclic ring of pheophytin followed by the non-enzymatic conversion of the unstable intermediate to pyropheophytin.
  • pheophorbidase A 28-29 kDa enzyme from Chenopodium album named pheophorbidase is reportedly capable of catalyzing an analogous reaction on pheophorbide, to produce the phytol-free derivative of pyropheophytin known as pyropheophorbide (see Figure 1). Pyropheophorbide is less polar than pheophorbide resulting in the pyropheophonbe having a decreased water solubility and an increased oil solubility compared with pheophorbide.
  • pyropheophytin can be more abundant than both pheophytin and chlorophyll in vegetable oils during processing (see Table 9 in volume 2.2. of Bailey's Industrial Oil and Fat Products (2005), 6 th edition, Ed. by Fereidoon Shahidi, John Wiley & Sons). This is partly because of the loss of magnesium from chlorophyll during harvest and storage of the plant material. If an extended heat treatment at 90°C or above is used, the amount of pyropheophytin in the oil is likely to increase and could be higher than the amount of pheophytin.
  • Chlorophyll levels are also reduced by heating of oil seeds before pressing and extraction as well as the oil degumming and alkali treatment during the refining process. It has also been observed that phospholipids in the oil can complex with magnesium and thus reduce the amount of chlorophyll. Thus chlorophyll is a relatively minor contaminant compared to pyropheophytin (and pheophytin) in many plant oils.
  • the present invention provides a process for treating a plant oil, comprising a step of contacting the oil with an enzyme which is capable of hydrolysing chlorophyll or a chlorophyll derivative, wherein the enzyme is contacted with the oil in the presence of up to 0.5% by weight water.
  • the enzyme is contacted with the oil in a one phase system, e.g. in an oil phase system with no observable aqueous phase formation and/or gum separation.
  • the plant oil is a crude plant oil.
  • the enzyme may be contacted with a crude oil before a step of degumming of the oil.
  • the plant oil is a degummed plant oil (e.g. an oil which has been treated to remove phophatides/phospholipids).
  • the enzyme may be contacted with a degummed oil, after a step of degumming of the oil.
  • the degumming step may comprise, for example, water degumming, acid degumming, enzymatic degumming, and/or total degumming/neutralisation (e.g. addition of an acid to the oil followed by neutralisation with an alkali).
  • the degumming step comprises water degumming.
  • the enzyme is contacted with the oil in the presence of less than 0.2% by weight lysophosholipid, for example less than 0.15%, less than 0.1% or less than 0.05% by weight, based on the total weight of oil.
  • the enzyme is contacted with the oil at a temperature of less than 80°C, less than 70°C, less than 60°C, less than 50°C, or less than 45°C.
  • the process may take place at 15°C to 75°C, 35°C to 65°C, 35°C to 45°C or 55°C to 65°C.
  • the enzyme is contacted with oil for at least 1 hour, at least 2 hours, at least 4 hours, at least 24 hours, at least 48 hours, at least 3 days, or at least 5 days.
  • the enzymatic incubation period may be 1 hour to 100 days, 1 hour to 30 days, 1 hour to 14 days, 1 to 24 hours, 1 to 4 hours, 1 to 14 days, 1 to 10 days, or 3 to 10 days.
  • the enzyme is contacted with the oil at a temperature of less than 50°C, less than 45°C or up to 40°C for at least 24 hours.
  • the oil is preferably a water-degummed oil. This step may take place, for example, during transport or shipping of the oil.
  • the enzyme is contacted with the oil at a temperature of 55°C to 65°C for 1 to 4 hours.
  • the oil is preferably a crude plant oil.
  • the enzyme comprises a chlorophyllase, pheophytinase, pyropheophytinase or pheophytin pheophorbide hydrolase.
  • the enzyme may comprise a polypeptide sequence as defined in any one of SEQ ID NOs: 1, 2, 4, 6 or 8 to 15, or a functional fragment or variant thereof.
  • the enzyme comprises a polypeptide sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1, 2, 4, 6 or 8 to 15, e.g. over at least 50 amino acid residues.
  • the enzyme is derived from Triticum spp. , preferably Triticum aestivum, e.g. the enzyme comprises the sequence of SEQ ID NO:2 or a sequence having at least 90% sequence identity thereto.
  • the invention provides a plant oil obtainable by a process as defined above.
  • chlorophyllases are active in a one phase system comprising up to 0.5% by weight water.
  • the enzyme may be contacted with the oil in the presence of a low concentration of water, thus avoiding the need for a specific incubation step in a two- phase (water/oil) system. This avoids the delay which may result from such a step, as well as the need for modification of typical oil refining facilities to accommodate the step.
  • the enzymatic incubation may take place during storage of e.g. a crude oil, or e.g. during transport or shipping of a water-degummed oil, such that there is no overall additional time requirement associated with the enzyme treatment.
  • the enzyme may typically be used at a low dose, thereby reducing cost.
  • the present demonstration of chlorophyllase activity in a one phase system is a surprising result.
  • Figure 1 shows the reactions involving chlorophyll and derivatives and enzymes used in the present invention.
  • Figure 2 shows the amino acid sequence of Arabidopsis thaliana chlorophyllase (SEQ ID NO:l).
  • Figure 3 shows the amino acid sequence of Triticum aestivum chlorophyllase (SEQ ID NO:2).
  • Figure 4 shows a nucleotide sequence encoding Triticum aestivum chlorophyllase (SEQ ID NO:3).
  • FIG. 5 shows the amino acid sequence of Chlamydomonas reinhardtii chlorophyllase (SEQ ID NO:4).
  • Figure 6 shows a nucleotide sequence encoding Chlamydomonas reinhardtii chlorophyllase (SEQ ID NO:5).
  • Figure 7 shows the amino acid sequence of a pheophytin pheophorbide hydrolase (PPH) from Arabidopsis thaliana (SEQ ID NO:6). A chloroplast transit peptide is shown in bold.
  • PPH pheophytin pheophorbide hydrolase
  • Figure 8 shows the nucleotide sequence of a cDNA from Arabidopsis thaliana encoding pheophytin pheophorbide hydrolase (SEQ ID NO:7).
  • the PPH of SEQ ID NO:6 is encoded by residues 173 to 1627 of SEQ ID NO:7.
  • Figure 9 shows the polypeptide sequence of Populus trichocarpa PPH (SEQ ID NO: 8).
  • Figure 10 shows the polypeptide sequence of Vitis vinifera PPH (SEQ ID NO:9).
  • Figure 11 shows the polypeptide sequence of Ricinus communis PPH (SEQ ID NO: 10).
  • Figure 12 shows the polypeptide sequence of Oryza sativa (jap nica cultivar-group) PPH (SEQ ID NO: 11).
  • Figure 13 shows the polypeptide sequence of Zea mays PPH (SEQ ID NO: 12).
  • Figure 14 shows the polypeptide sequence of Nicotiana tabacum PPH (SEQ ID NO: 13).
  • Figure 15 shows the polypeptide sequence of Oryza sativa Japonica Group PPH (SEQ ID NO: 14).
  • Figure 16 shows (a) the polypeptide sequence of Physcomitrella patens subsp. patens PPH (SEQ ID NO: 15)
  • Figure 17 shows schematically the fusion of the wheat (Triticum aestivum) chlorophyllase gene to the aprE signal sequence.
  • Figure 18 shows schematically the plasmid pBN-TRI CHL containing the wheat (Triticum aestivum) chlorophyllase gene.
  • Figure 19 shows schematically the fusion of the Chlamydomonas reinhardtii chlorophyllase gene to the aprE signal sequence.
  • Figure 20 shows schematically the plasmid pBN-CHL_CHL containing the Chlamydomonas reinhardtii chlorophyllase gene.
  • Figure 21 shows the effect of water content on Triticum chlorophyllase (powder) activity on pheophytin.
  • Figure 22 shows the effect of water content on Triticum chlorophyllase (powder) activity on pyropheophytin.
  • Figure 23 shows the amino acid sequence of a mutant Aeromonas salmonicida mature lipid acyltransferase (GCAT) with a mutation of Asn80Asp after undergoing post- translational modification (SEQ ID No. 23)
  • Figure 24 shows the effect of chlorophyllase on pheophytin in water degumrned oil, in a low water environment.
  • Figure 25 shows the effect of chlorophyllase on pyropheophytin in water degummed oil in a low water environment.
  • Figure 26 is a diagrammatic representation of an oil refining process according to an embodiment of the present invention.
  • the present invention relates to a process for treating a plant oil.
  • the process is used to remove chlorophyll and/or chlorophyll derivatives from the oil, or to reduce the level of chlorophyll and/or chlorophyll derivatives in the oil, for instance where the chlorophyll and/or chlorophyll derivatives are present as a contaminant.
  • chlororophyll derivative it is typically meant compounds which comprise both a porphyrin (chlorin) ring and a phytol group (tail), including magnesium-free phytol- containing derivatives such as pheophytin and pyropheophytin. Chlorophyll and (phytol-containing) chlorophyll derivatives are typically greenish is colour, as a result of the porphyrin (chlorin) ring present in the molecule. Loss of magnesium from the porphyrin ring means that pheophytin and pyropheophytin are more brownish in colour than chlorophyll.
  • the present process may be performed in order to remove or reduce the green or brown colouring present in the oil. Accordingly the present process may be referred to as a bleaching or de-colorizing process.
  • Enzymes used in the process may hydrolyse chlorophyll and phytol-containing chlorophyll derivatives to cleave the phytol tail from the chlorin ring. Hydrolysis of chlorophyll and chlorophyll derivatives typically results in compounds such as chlorophyllide, pheophorbide and pyropheophorbide which are phytol-free derivatives of chlorophyll.
  • the process may further comprise a step of removing or reducing the level of phytol-free chlorophyll derivatives in the oil.
  • chlorophyll or chlorophyll derivative may be either a or b forms.
  • chlororophyll includes chlorophyll a and chlorophyll b. In a similar way both a and b forms are covered when referring to pheophytin, pyropheophytin, chlorophyllide, pheophorbide and pyropheophorbide.
  • Any plant oil may be treated according to the present process, in order to remove undesirable contamination by chlorophyll and/or chlorophyll derivatives.
  • the oil may be derived from any type of plant, and from any part of a plant, including whole plants, leaves, stems, flowers, roots, plant protoplasts, seeds and plant cells and progeny of same.
  • the class of plants from which products can be treated in the method of the invention includes higher plants, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous states.
  • the oil may comprise a vegetable oil, including oils processed from oil seeds or oil fruits (e.g. seed oils such as canola (rapeseed) oil and fruit oils such as palm).
  • suitable oils include rice bran, soy, canola (rape seed), palm, olive, cottonseed, corn, palm kernel, coconut, peanut, sesame, Moringa or sunflower.
  • the process of the invention can be used in conjunction with methods for processing essential oils, e.g., those from fruit seed oils, e.g. grapeseed, apricot, borage, etc.
  • the process of the invention can be used in conjunction with methods for processing high phosphorus oils (e.g. a soy bean oil).
  • the chlorophyll and/or chlorophyll derivatives may be present in the oil naturally, as a contaminant, or as an undesired component in a processed product.
  • the chlorophyll and/or chlorophyll derivatives e.g. chlorophyll, pheophytin and/or pyropheophytin
  • chlorophyll, pheophytin and/or pyropheophytin may be present as a natural contaminant in the oil at a concentration of 0.001 to 1000 mg/kg (0.001 to 1000 ppm, 10 "7 to 10 "1 wt %), based on the total weight of the oil.
  • the chlorophyll and/or chlorophyll derivatives may be present in the oil at a concentration of 0.1 to 100, 0.5 to 50, 1 to 50, 1 to 30 or 1 to 10 mg/kg, based on the total weight of the oil.
  • chlorophyllide, pyropheophorbide and/or pyropheophorbide may be present at any level in the oil.
  • chlorophyllide, pyropheophorbide and/or pyropheophorbide may be present in the oil, either before or after treatment with an enzyme according to the method of the present invention, at a concentration of 0.001 to 1000 mg/kg (0.001 to 1000 ppm, 10 "7 to 10 "1 wt %), based on the total weight of the oil.
  • the chlorophyllide, pyropheophorbide and/or pyropheophorbide may be present in the composition at a concentration of 0.1 to 100, 0.5 to 50, 1 to 50, 1 to 30 or 1 to 10 mg/kg, based on the total weight of the composition.
  • the process of the present invention comprises a step of contacting the oil with an enzyme which is capable of hydrolysing chlorophyll or a chlorophyll derivative.
  • an enzyme capable of hydrolysing chlorophyll or a chlorophyll derivative.
  • hydrolyzing chlorophyll or a chlorophyll derivative means hydrolysing an ester bond in chlorophyll or a (phytol-containing) chlorophyll derivative, e.g. to cleave a phytol group from the chlorin ring in the chlorophyll or chlorophyll derivative.
  • the enzyme typically has an esterase or hydrolase activity.
  • the enzyme has esterase or hydrolase activity in an oil phase, and optionally also in an aqueous phase.
  • the enzyme may, for example, be a chlorophyllase, pheophytinase or pyropheophytinase.
  • the enzyme is capable of hydrolysing at least one, at least two or all three of chlorophyll, pheophytin and pyropheophytin.
  • the enzyme has chlorophyllase, pheophytinase and pyropheophytinase activity.
  • two or more enzymes may be used in the method, each enzyme having a different substrate specificity.
  • the method may comprise the combined use of two or three enzymes selected from a chlorophyllase, a pheophytinase and a pyropheophytinase.
  • Any polypeptide having an activity that can hydrolyse chlorophyll or a chlorophyll derivative can be used as the enzyme in the process of the invention.
  • enzyme it is intended to encompass any polypeptide having hydrolytic activity on chlorophyll or a chlorophyll derivative, including e.g. enzyme fragments, etc.
  • Any isolated, recombinant or synthetic or chimeric (or a combination of synthetic and recombinant) polypeptide can be used.
  • Enzyme (chlorophyllase, pheophytinase or pyropheophytinase) activity assay
  • Hydrolytic activity on chlorophyll or a chlorophyll derivative may be detected using any suitable assay technique, for example based on an assay described herein.
  • hydrolytic activity may be detected using fluorescence-based techniques.
  • a polypeptide to be tested for hydrolytic activity on chlorophyll or a chlorophyll derivative is incubated in the presence of a substrate, and product or substrate levels are monitored by fluorescence measurement.
  • Suitable substrates include e.g. chlorophyll, pheophytin and/or pyropheophytin.
  • Products which may be detected include chlorophyllide, pheophorbide, pyropheophorbide and/or phytol.
  • a suitable assay may be based on HPLC detection and quantitation of substrate or product levels following addition of a putative enzyme, e.g. based on the techniques described below.
  • the assay may be performed as described in Hornero-Mendez et al. (2005), Food Research International 38(8-9): 1067- 1072. h another embodiment, the following assay may be used:
  • One unit of enzyme activity is defined as the amount of enzyme which hydrolyzes one micromole of substrate (e.g. chlorophyll, pheophytin or pyropheophytin) per minute at 40°C, e.g. in an assay method as described herein.
  • substrate e.g. chlorophyll, pheophytin or pyropheophytin
  • the enzyme used in the present method has chlorophyllase, pheophytinase and/or pyropheophytinase 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 the units of activity per gram of the purified enzyme, e.g. as determined by an assay method described herein.
  • hydrolytic activity on chlorophyll or a chlorophyll derivative may be determined using a method as described in EP10159327.5.
  • the enzyme is capable of hydrolyzing at least chlorophyll.
  • Any polypeptide that catalyses the hydrolysis of a chlorophyll ester bond to yield chlorophyllide and phytol can be used in the process.
  • a chlorophyllase, chlase or chlorophyll chlorophyllido-hydrolyase or polypeptide having a similar activity e.g., chlorophyll-chlorophyllido hydrolase 1 or chlase 1, or, chlorophyll-chlorophyllido hydrolase 2 or chlase 2, see, e.g. NCBI P59677-1 and P59678, respectively
  • NCBI P59677-1 and P59678 see, e.g. NCBI P59677-1 and P59678, respectively
  • the enzyme is a chlorophyllase classified under the Enzyme Nomenclature classification (E.G. 3.1.1.14). Any isolated, recombinant or synthetic or chimeric (a combination of synthetic and recombinant) polypeptide (e.g., enzyme or catalytic antibody) can be used, see e.g. Marchler-Bauer (2003) Nucleic Acids Res. 31 : 383-387.
  • the chlorophyllase may be an enzyme as described in WO 0229022 or WO 2006009676.
  • the Arabidopsis thaliana chlorophyllase can be used as described, e.g. in NCBI entry NM_123753.
  • the chlorophyllase may be a polypeptide comprising the sequence of SEQ ID NO:l (see Figure 2).
  • the chlorophyllase is derived from algae, e.g. from Phaeodactylum tricornutum.
  • the chlorophyllase is derived from wheat, e.g. from Triticum sp., especially from Triticum aestivum.
  • the chlorophyllase may be polypeptide comprising the sequence of SEQ ID NO:2 (see Figure 3), or may be encoded by the nucleotide sequence of SEQ ID NO:3 (see Figure 4).
  • the chlorophyllase is derived from Chlamydomonas sp., especially from Chlamydomonas reinhardtii.
  • the chlorophyllase may be a polypeptide comprising the sequence of SEQ ID NO:4 (see Figure 5), or may be encoded by the nucleotide sequence of SEQ ID NO:5 (see Figure 6).
  • the enzyme is capable of hydrolyzing pheophytin and pyropheophytin.
  • the enzyme may be pheophytinase or pheophytin pheophorbide hydrolase (PPH), e.g. an enzyme as described in Schelbert et al., The Plant Cell 21 :767-785 (2009).
  • PPH and related enzymes are capable of hydrolyzing pyropheophytin in addition to pheophytin.
  • PPH is inactive on chlorophyll.
  • PPH orthologs are commonly present in eukaryotic photosynthesizing organisms.
  • PPHs represent a defined sub-group of ⁇ / ⁇ hydrolases which are phylogenetically distinct from chlorophyllases, the two groups being distinguished in terms of sequence homology and substrates.
  • the enzyme may be any known PPH derived from any species or a functional variant or fragment thereof or may be derived from any known PPH enzyme.
  • the enzyme is a PPH from Arabidopsis thaliana, e.g. a polypeptide comprising the amino acid sequence of SEQ ID NO:6 (see Figure 7), or a polypeptide encoded by the nucleotide sequence of SEQ ID NO:7 (see Figure 8, NCBI accession no. NPJ 96884, GenBank ID No. 15240707), or a functional variant or fragment thereof.
  • the enzyme may be a PPH derived from any one of the following species: Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera, Oryza sativa, Zea mays, Nicotiana tabacum, Ostreococcus lucimarinus, Ostreococcus taurii, Physcomitrella patens, Phaeodactylum tricornutum, Chlamydomonas reinhardtii, or Micromonas sp. RCC299.
  • the enzyme may be a polypeptide comprising an amino acid sequence, or encoded by a nucleotide sequence, defined in one of the following database entries shown in Table 1 , or a functional fragment or variant thereof:
  • the enzyme may be a polypeptide as defined in any of SEQ ID NO:s 8 to 15 ( Figures 9 to 16), or a functional fragment or variant thereof.
  • variants and fragments of known sequences which hydrolyse chlorophyll or a chlorophyll derivative may also be employed in the present invention.
  • functionar it is meant that the fragment or variant retains a detectable hydrolytic activity on chlorophyll or a chlorophyll derivative.
  • variants and fragments show homology to a known chlorophyllase, pheophytinase or pyropheophytinase sequence, e.g.
  • the percentage of sequence identity may be determined by analysis with a sequence comparison algorithm or by a visual inspection.
  • the sequence comparison algorithm is a BLAST algorithm, e.g., a BLAST version 2.2.2 algorithm.
  • enzymes having chlorophyllase, pheophytinase and/or pyropheophytinase activity suitable for use in the process may be identified by determining the presence of conserved sequence motifs present e.g. in known chlorophyllase, pheophytinase or pyropheophytinase sequences.
  • conserved sequence motifs found in PPH enzymes include the following: LPGFGVG (SEQ ID NO: 16), DFLGQG (SEQ ID NO:17), GNSLGG (SEQ ID NO:18), LVKGVTLLNATPFW (SEQ ID NO: 19), HPAA (SEQ ID NO:20), EDPW (SEQ ID NO:21), and SPAGHCPH (SEQ ID NO:22).
  • an enzyme for use in the present invention may comprise one or more of these sequences.
  • the GNSLGG (SEQ ID NO: 18) motif contains an active site serine residue.
  • Polypeptide sequences having suitable activity may be identified by searching genome databases, e.g. the microbiome metagenome database (JGI-DOE, USA), for the presence of these motifs.
  • Enzymes for use in the present invention may be isolated from their natural sources or may be, for example, produced using recombinant DNA techniques. Nucleotide sequences encoding polypeptides having chlorophyllase, pheophytinase and/or pyropheophytinase activity may be isolated or constructed and used to produce the corresponding polypeptides.
  • a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the polypeptide. If the amino acid sequence of the polypeptide is known, labeled oligonucleotide probes may be synthesised and used to identify polypeptide-encoding clones from the genomic library prepared from the organism. Alternatively, a labelled oligonucleotide probe containing sequences homologous to another known polypeptide gene could be used to identify polypeptide-encoding clones. In the latter case, hybridisation and washing conditions of lower stringency are used.
  • polypeptide-encoding clones could be identified by inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming enzyme- negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing an enzyme inhibited by the polypeptide, thereby allowing clones expressing the polypeptide to be identified.
  • an expression vector such as a plasmid, transforming enzyme- negative bacteria with the resulting genomic DNA library
  • the nucleotide sequence encoding the polypeptide may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S.L. et al (1981) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al (1984) EMBO J. 3, p 801-805.
  • the phosphoroamidite method oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, li gated and cloned in appropriate vectors.
  • the nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence.
  • the DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or in Saiki R et al (Science (1988) 239, pp 487-491).
  • nucleotide sequence refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homologues, fragments and derivatives thereof (such as portions thereof).
  • the nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or antisense strand.
  • nucleotide sequence encoding a polypeptide having chlorophyllase, pheophytinase and/or pyropheophytinase activity is prepared using recombinant DNA techniques.
  • the nucleotide sequence could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).
  • an enzyme-encoding nucleotide sequence has been isolated, or a putative enzyme- encoding nucleotide sequence has been identified, it may be desirable to modify the selected nucleotide sequence, for example it may be desirable to mutate the sequence in order to prepare an enzyme in accordance with the present invention.
  • Mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites. A suitable method is disclosed in Morinaga et al (Biotechnology (1984) 2, p646-649). Another method of introducing mutations into enzyme-encoding nucleotide sequences is described in Nelson and Long (Analytical Biochemistry (1989), 180, p 147-151).
  • EP 0 583 265 refers to methods of optimising PCR based mutagenesis, which can also be combined with the use of mutagenic DNA analogues such as those described in EP 0 866 796.
  • Error prone PCR technologies are suitable for the production of variants of enzymes which hydrolyse chlorophyll and/or chlorophyll derivatives with preferred characteristics.
  • WO0206457 refers to molecular evolution of lipases.
  • a third method to obtain novel sequences is to fragment non-identical nucleotide sequences, either by using any number of restriction enzymes or an enzyme such as Dnase I, and reassembling full nucleotide sequences coding for functional proteins. Alternatively one can use one or multiple non-identical nucleotide sequences and introduce mutations during the reassembly of the full nucleotide sequence.
  • DNA shuffling and family shuffling technologies are suitable for the production of variants of enzymes with preferred characteristics. Suitable methods for performing 'shuffling' can be found in EP0752008, EPl 138763, EPl 103606. Shuffling can also be combined with other forms of DNA mutagenesis as described in US 6,180,406 and WO 01/34835.
  • mutations or natural variants of a polynucleotide sequence can be recombined with either the wild type or other mutations or natural variants to produce new variants.
  • Such new variants can also be screened for improved functionality of the encoded polypeptide.
  • an enzyme may be altered to improve the functionality of the enzyme.
  • a nucleotide sequence encoding an enzyme e.g. a chlorophyllase, pheophytinase and/or pyropheophytinase
  • an enzyme e.g. a chlorophyllase, pheophytinase and/or pyropheophytinase
  • the variant enzyme may contain at least one amino acid substitution, deletion or addition, when compared to a parental enzyme.
  • Variant enzymes retain at least 1%, 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50 %, 60%, 70%, 80%, 90%, 95%, 97%, or 99% identity with the parent enzyme.
  • Suitable parent enzymes may include any enzyme with hydrolytic activity on chlorophyll and/or a chlorophyll derivative.
  • the present invention also encompasses the use of amino acid sequences encoded by a nucleotide sequence which encodes a chlorophyllase, pheophytinase or pyropheophytinase for use in any one of the methods and/or uses of the present invention.
  • amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”.
  • amino acid sequence is synonymous with the term “peptide”.
  • the amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques. Suitably, the amino acid sequences may be obtained from the isolated polypeptides taught herein by standard techniques.
  • One suitable method for determining amino acid sequences from isolated polypeptides is as follows. Purified polypeptide may be freeze-dried and 100 ⁇ g of the freeze-dried material may be dissolved in 50 ⁇ of a mixture of 8 M urea and 0.4 M ammonium hydrogen carbonate, pH 8.4. The dissolved protein may be denatured and reduced for 15 minutes at 50°C following overlay with nitrogen and addition of 5 ⁇ of 45 mM dithiothreitol. After cooling to room temperature, 5 ⁇ of 100 mM iodoacetamide may be added for the cysteine residues to be derivatized for 15 minutes at room temperature in the dark under nitrogen.
  • homologue means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences.
  • the term “homology” can be equated with "identity”.
  • the homologous amino acid sequence and/or nucleotide sequence should provide and/or encode a polypeptide which retains the functional activity and/or enhances the activity of the enzyme.
  • a homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the subject sequence.
  • the homologues will comprise the same active sites etc. as the subject amino acid sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • a homologous sequence is taken to include a nucleotide sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to a nucleotide sequence encoding a polypeptide of the present invention (the subject sequence).
  • the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences. % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • a suitable computer program for carrying out such an alignment is the Vector NTI AdvanceTM 11 (Invitrogen Corp.).
  • Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al 1999 Short Protocols in Molecular Biology, 4th Ed - Chapter 18), and FASTA (Altschul et al 1990 J. Mol. Biol. 403-410). Both BLAST and FASTA are available for offline and online searching (see Ausubel et al 1999, pages 7-58 to 7-60). However, for some applications, it is preferred to use the Vector NTI AdvanceTM 11 program.
  • a new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; and FEMS Microbiol Lett 1999 177(1): 187-8.).
  • % homology can be measured in terms of identity
  • the alignment process itself is typically not based on an all-or-nothing pair comparison.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • Vector NTI programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the default values for the Vector NTI AdvanceTM 11 package.
  • percentage homologies may be calculated using the multiple alignment feature in Vector NTI AdvanceTM 11 (Invitrogen Corp.), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).
  • CLUSTAL Higgins DG & Sharp PM (1988), Gene 73(1), 237-244
  • the default parameters for the programme are used for pairwise alignment.
  • the following parameters are the current default parameters for pairwise alignment for BLAST 2: FOR BLAST2 DNA PROTEIN
  • sequence identity for the nucleotide sequences and/or amino acid sequences may be determined using BLAST2 (blastn) with the scoring parameters set as defined above.
  • the degree of identity is based on the number of sequence elements which are the same.
  • the degree of identity in accordance with the present invention for amino acid sequences may be suitably determined by means of computer programs known in the art such as Vector NTI AdvanceTM 11 (Invitrogen Corp.).
  • the scoring parameters used are preferably BLOSUM62 with Gap existence penalty of 1 land Gap extension penalty of 1.
  • the degree of identity with regard to a nucleotide sequence is determined over at least 20 contiguous nucleotides, preferably over at least 30 contiguous nucleotides, preferably over at least 40 contiguous nucleotides, preferably over at least 50 contiguous nucleotides, preferably over at least 60 contiguous nucleotides, preferably over at least 100 contiguous nucleotides.
  • the degree of identity with regard to a nucleotide sequence may be determined over the whole sequence.
  • sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.
  • 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 having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • AROMATIC H F W Y The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e.
  • unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenyl glycine. Replacements may also be made by unnatural amino acids.
  • Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or ⁇ - alanine residues.
  • alkyl groups such as methyl, ethyl or propyl groups
  • amino acid spacers such as glycine or ⁇ - alanine residues.
  • a further form of variation involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art.
  • the peptoid form is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the residue's nitrogen atom rather than the a-carbon.
  • Nucleotide sequences for use in the present invention or encoding a polypeptide having the specific properties defined herein may include within them synthetic or modified nucleotides.
  • a number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences.
  • the present invention also encompasses the use of nucleotide sequences that are complementary to the sequences discussed herein, or any derivative, fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.
  • Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways.
  • Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations.
  • other viral/bacterial, or cellular homologues particularly cellular homologues found in plant cells may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein.
  • Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other plant species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.
  • Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention.
  • conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
  • the primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
  • polynucleotides may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon sequence changes are required to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction polypeptide recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
  • Polynucleotides (nucleotide sequences) of the invention may be used to produce a primer, e.g. a PGR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or nonradioactive labels, or the polynucleotides may be cloned into vectors.
  • a primer e.g. a PGR primer
  • a primer for an alternative amplification reaction e.g. labelled with a revealing label by conventional means using radioactive or nonradioactive labels
  • the polynucleotides may be cloned into vectors.
  • Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
  • Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
  • primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the enzyme sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from a plant cell, performing a polymerase chain reaction under conditions which bring about 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.
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
  • Enzyme formulation and dosage Enzymes used in the methods of the invention can be formulated or modified, e.g., chemically modified, to enhance oil solubility, stability, activity or for immobilization.
  • enzymes used in the methods of the invention can be formulated to be amphipathic or more lipophilic.
  • enzymes used in the methods of the invention can be encapsulated, e.g., in liposomes or gels, e.g., alginate hydrogels or alginate beads or equivalents.
  • Enzymes used in the methods of the invention can be formulated in micellar systems, e.g., a ternary micellar (TMS) or reverse micellar system (RMS) medium.
  • Enzymes used in the methods of the invention can be formulated as described in Yi (2002) J. of Molecular Catalysis B: Enzymatic, Vol. 19, pgs 319-325.
  • the enzymatic reactions of the methods of the invention e.g. the step of contacting the oil with an enzyme which hydrolyses chlorophyll or a chlorophyll derivative, can be done in one reaction vessel or multiple vessels.
  • the enzymatic reactions of the methods of the invention are done in a vegetable oil refining unit or plant.
  • the method of the invention can be practiced with immobilized enzymes, e.g. an immobilized chlorophyllase, pheophytinase and/or pyropheophytinase.
  • the enzyme can be immobilized on any organic or inorganic support.
  • Exemplary inorganic supports include alumina, celite, Dowex-1 -chloride, glass beads and silica gel.
  • Exemplary organic supports include DEAE-cellulose, alginate hydrogels or alginate beads or equivalents.
  • immobilization of the enzyme can be optimized by physical adsorption on to the inorganic support.
  • Enzymes used to practice the invention can be immobilized in different media, including water, Tris-HCl buffer solution and a ternary micellar system containing Tris-HCl buffer solution, hexane and surfactant.
  • the enzyme can be immobilized to any type of substrate, e.g. filters, fibers, columns, beads, colloids, gels, hydrogels, meshes and the like.
  • the enzyme may be dosed into the oil in any suitable amount.
  • the enzyme may be dosed in a range of about 0.001 to lOU/g of the composition, preferably 0.01 to 1 U/g, e.g. 0.01 to 0.1 U/g of the oil.
  • One unit is defined as the amount of enzyme which hydrolyses 1 ⁇ of substrate (e.g. chlorophyll, pheophytin and/or pyropheophytin) per minute at 40 °C, e.g. under assay conditions as described in J. Biol. Chem. (1961) 236: 2544-2547.
  • the enzyme dosage is less than 1 U/g oil, more preferably less than 0.5 U/g oil, more preferably less than 0.2 U/g oil.
  • Such low doses of the enzyme may advantageously be used in combination with extended incubation times and/or low temperature incubations with the oil, e.g. in embodiments where the enzyme is contacted with a water degummed oil during transport or shipping.
  • the enzyme is contacted with a crude plant oil.
  • the enzyme is contacted with a degummed oil.
  • the incubation with the enzyme may be performed before or after a step of degumming the oil.
  • Crude and degummed oils may be distinguished from one another in terms of their phospholipid content.
  • the phospholipid content of plant oils varies according to the particular source and nature of the oil and the stage of the refining process.
  • the phospholipid content of crude plant oils may be up to 5% by weight at the start of the process, but following a water degumming step the phospholipid content typically falls to 1% by weight or below, e.g. around 0.3 % by weight.
  • an enzymatic degumming step e.g. using a phospholipase
  • a total degumming step e.g.
  • the phospholipid content may fall much lower, for example below 0.1% or even below 0.01% by weight based on the total weight of the oil.
  • Typical phospholipid contents in % by weight of some common oils are shown below:
  • oils may be determined using standard methods. For example, phospholipid levels in oils may be determined as described in J. Amer. Oil. Chem. Soc. 58, 561 (1981). In one embodiment phospholipid levels may be determined by thin- layer chromatography (TLC) analysis, e.g. as described in WO 2006/008508 or WO 03/100044.
  • TLC thin- layer chromatography
  • Phospholipid levels in oil can also be determined by (a) AOCS Recommended Practice Ca 19-86 (reapproved 2009), "Phospholipids in Vegetable Oils Nephelometric Method” or (b) AOCS Official Method Ca 20-99 (reapproved 2009), "Analysis for Phosphorus in Oil by Inductively Coupled Plasma Optical Emission Spectroscopy".
  • the crude oil is an oil comprising at least 0.5%, at least 1.0% or at least 2% by weight phospholipid.
  • the oil is a water degummed plant oil comprising 0.1 to 1% by weight phospholipid.
  • the enzyme is contacted with the oil at a time when a concentration of lysophospholipid in the oil is as low as possible.
  • the enzyme may be contacted with the oil in the presence of less than 0.2% by weight lysophosholipid.
  • lysophosholipid it is meant that the lysophospholipid content in the oil is less than 0.2%» by weight, e.g. based on the total weight of the oil composition, for at least a part of a time during which the enzyme is incubated with the oil (e.g. at least at a time when the enzyme is added to the oil).
  • the lysophospholipid content in the oil may be any value below 0.2% by weight, including zero.
  • Lysophospholipids may be produced during oil processing by cleavage of an acyl (fatty acid) chain from phospholipids, leaving a single acyl chain, a phosphate group, optionally a headgroup and a free alcohol attached to the glyceryl moiety.
  • Enzymes used in degumming such as phospholipases (in particular phospholipase Al and A2) and acyltransferases may generate lysophospholipids in the oil.
  • the enzyme which hydrolyses chlorophyll or a chlorophyll derivative may be contacted with the oil before the enzymatic degumming step.
  • a higher dose or extended incubation time of the chlorophyllase or related enzyme may be required, in order to overcome any reduction in activity due to the presence of lysophospholipids.
  • a lysophospholipase may be used in combination with a phospholipase or acyltransferase in the degumming step.
  • Lysophospholipases (EC 3.1.1.5) are enzymes that can hydrolyze lysophospholipids to release fatty acid. Use of a lysophospholipase may help to reduce the production of lysophospholipids in the oil during the degumming step, e.g. to maintain the lysophospholipid content of the oil below about 0.2% by weight.
  • Suitable lysophospholipases are disclosed, for example, in Masuda et al., Eur. J. Biochem., 202,783-787 (1991); WO 98/31790; WO 01/27251 and WO 2008/040465.
  • Phospholipase C is another enzyme which may be used in degumming. Phospholipase C cleaves phospholipids between the glyceryl and phosphate moieties, leaving diacylglycerol and a phosphate group (attached to a headgroup if present). Thus in contrast to phospholipase Al and A2, phospholipase C does not produce lysophospholipids.
  • the lysophospholipid content of the oil is less than 0.2%, less than 0.15%, less than 0.1% or less than 0.05% by weight, based on the total weight of oil. In general, concentrations of lysophospholipid which are as low as possible are desirable.
  • Lysophospholipids which may be present in the oil include lysophosphatidylcholine (LPC), lysophosphatidylinositol (LPI), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS) and lysophosphatidic acid (LP A). It is particularly preferred that the level of LPC and LPE in the oil is as low as possible, h preferred embodiments, the concentration of LPC and/or LPE is less than 0.2%, less than 0.15%, less than 0.1 % or less than 0.05% by weight, based on the total weight of oil.
  • the lysophospholipid content of oils maybe determined using standard methods, e.g.
  • the oil may be incubated (or admixed) with the enzyme between about 5°C to and about 100°C, more preferably between 10°C to about 90°C, more preferably between about 15°C to about 80°C, more preferably between about 20°C to about 75°C.
  • pheophytin is decomposed to pyropheophytin, which is generally less preferred because some chlorophyllases are less active on pyropheophytin compared to pheophytin.
  • the chlorophyllase degradation product of pyropheophytin, pyropheophorbide is less water soluble compared to pheophorbide and thus more difficult to remove from the oil afterwards.
  • the enzymatic reaction rate is increased at higher temperatures but it is favourable to keep the conversion of pheophytin to pyropheophytin to a minimum.
  • the oil is incubated with the enzyme at below about 80°C, preferably below about 70°C, preferably at about 68°C or below, preferably at about 65 °C or below, in order to reduce the amount of conversion to pyropheophytin.
  • the reaction time is relatively short (e.g. less than 24 hours, typically less than about 4 hours)
  • preferred temperature ranges for the incubation of the enzyme with the oil include about 50°C to below about 70°C, about 50°C to about 65 °C and about 55°C to about 65 °C.
  • a lower temperature may be used (typically in combination with a longer reaction time).
  • the temperature is typically below 50°C, below about 45°C, below about 40°C, below about 35°C, below about 30°C, or below about 25°C.
  • the enzyme may be contacted with the oil at ambient temperature, e.g. 15 to 25°C.
  • the temperature of the oil may be at the desired reaction temperature when the enzyme is admixed therewith.
  • the oil may be heated and/or cooled to the desired temperature before and/or during enzyme addition. Therefore in one embodiment it is envisaged that a further step of the process according to the present invention may be the cooling and/or heating of the oil.
  • the reaction time (i.e. the time period in which the enzyme is incubated with the oil), preferably with agitation, is for a sufficient period of time to allow hydrolysis of chlorophyll and chlorophyll derivatives, e.g. to form phytol and chlorophyllide, pheophorbide and/or pyropheophorbide.
  • the reaction time may be at least about 1 minute, more preferable at least about 5 minutes, more preferably at least about 10 minutes.
  • the reaction time may be between about 15 minutes to about 6 hours, preferably between about 15 minutes to about 60 minutes, preferably about 30 to about 120 minutes.
  • the reaction time may up to 6 hours, or up to 24 hours.
  • the reaction temperature may be extended, for instance to include the duration of a transport or shipping step of the oil.
  • the reaction time may be at least 24 hours, at least 48 hours, at least 3 days, at least 5 days, at least 10 days, at least 20 days or at least 50 days, e.g. 1 to 50 days, 1 to 20 days, or 3 to 10 days.
  • the step of contacting the enzyme with the oil is typically performed in the presence of up to 0.5% by weight of water, e.g. based on the total weight of oil.
  • water content may be less than 0.5% by weight, or less than 0.49%, 0.48%, 0.47%, 0.46%, 0.45%, 0.4%, 0.3%, or 0.2% by weight.
  • the water content is at least 0.1% by weight, more preferably at least 0.2%, 0.3% or 0.4% by weight.
  • preferred water content ranges include 0.1 to 0.5%, 0.1 to 0.49%, 0.1 to 0.48%, 0.1 to 0.47%, 0.1 to 0.46%, 0.1 to 0.45%, 0.1 to 0.4%, 0.1 to 0.3%, 0.1 to 0.2%, 0.2 to 0.5%, 0.2 to 0.49%, 0.2 to 0.48%, 0.2 to 0.47%, 0.2 to 0.46%, 0.2 to 0.45%, 0.2 to 0.4%, 0.2 to 0.3%, 0.3 to 0.5%, 0.3 to 0.49%, 0.3 to 0.48%, 0.3 to 0.47%, 0.3 to 0.46%, 0.3 to 0.45%, 0.3 to 0.4%, 0.4 to 0.5%, 0.40 to 0.49%, 0.40 to 0.48%, 0.40 to 0.47%, 0.40 to 0.46% and 0.40 to 0.45% by weight.
  • the enzyme is contacted with the oil in a one phase system.
  • a one phase system By this it is meant that the step takes place in a single phase mixture, comprising an oil phase but no distinguishable aqueous phase.
  • Two phase formation may be readily observed by the naked eye when a higher water content is used. At such higher water contents (typically greater than 0.5%), phase separation into oil and aqueous phases and/or gum separation is usually seen. pH
  • the process is carried out between about pH 4.0 and about pH 10.0, more preferably between about pH 5.0 and about pH 10.0, more preferably between about pH 6.0 and about pH 10.0, more preferably between about pH 5.0 and about pH 7.0, more preferably between about pH 5.0 and about pH 7.0, more preferably between about pH 6.5 and about pH 7.0, e.g. at about pH 7.0 (i.e. neutral pH).
  • the process is carried out between about pH 5.5 and pH 6.0.
  • the treated liquid e.g. oil
  • an appropriate means such as a centrifugal separator and the processed oil is obtained.
  • the processed oil can be additionally washed with water, an alkali or organic or inorganic acid such as, e.g., acetic acid, citric acid, phosphoric acid, succinic acid, and the like, or with salt solutions.
  • Chlorophyll and/or chlorophyll derivative removal typically reduces the level of chlorophyll and/or chlorophyll derivatives in the oil.
  • the process may reduce the concentration of chlorophyll, pheophytin and/or pyropheophytin by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%, compared to the concentration of chlorophyll, pheophytin and/or pyropheophytin (by weight) present in the oil before treatment.
  • the concentration of chlorophyll and/or chlorophyll derivatives in the oil after treatment may be less than 100, less than 50, less than 30, less than 10, less than 5, less than 1, less than 0.5, less than 0.1 mg kg or less than 0.02 mg/kg, based on the total weight of the oil.
  • oil is extracted in hexane, the crude vegetable oil is degummed, optionally caustic neutralized, bleached using, e.g. clay adsorption with subsequent clay disposal, and deodorized to produce refined, bleached and deodorized or RBD oil (see Figure 26).
  • the need for the degumming step depends on phosphorus content and other factors.
  • the process of the present invention can be used in conjunction with processes based on extraction with hexane and/or enzyme assisted oil extraction (see Journal of Americal Oil Chemists' Society (2006), 83 (11), 973-979). In general, the process of the invention may be performed using oil processing steps as described in Bailey's Industrial Oil and Fat Products (2005), 6 th edition, Ed. by Fereidoon Shahidi, John Wiley & Sons.
  • an enzymatic reaction involving application of the enzyme capable of hydrolyzing chlorophyll or a chlorophyll derivative is preferably performed at specific stages in this process. Preferred stages of the process for using the enzyme according to the present process are shown in Figure 26.
  • the enzyme is preferably contacted with the oil before the degumming step.
  • the enzyme may be contacted with the oil after a water degumming step. The enzyme is typically contacted with the oil before degumming is complete (e.g. before a caustic neutralization step).
  • the enzyme may be contacted with the oil after water degumming (e.g.
  • the enzyme is added to water-degummed oil), but preferably the enzymatic hydrolysis of chlorophyll and chlorophyll derivatives is performed before a total degumming step, e.g. before addition of acid and caustic neutralization. This is shown in Figure 26.
  • the enzyme may be added after partial degumming of the oil.
  • Further processing steps after treatment with the enzyme, may assist in removal of the products of enzymatic hydrolysis of chlorophyll and/or chlorophyll derivatives. For instance, further processing steps may remove chlorophyllide, pheophorbide, pyropheophorbide and/or phytol.
  • the degumming step in oil refining serves to separate phosphatides by the addition of water.
  • the material precipitated by degumming is separated and further processed to mixtures of lecithins.
  • the commercial lecithins such as soybean lecithin and sunflower lecithin, are semi-solid or very viscous materials. They consist of a mixture of polar lipids, primarily phospholipids such as phosphatidylcholine with a minor component of triglycerides.
  • the term "degumming” means the refining of oil by removing phospholipids from the oil.
  • degumming may comprise a step of converting phosphatides (such as lecithin and phospholipids) into hydratable phosphatides.
  • the process of the invention can be used with any degumming procedure, particularly in embodiments where the chlorophyll- or chlorophyll derivative-hydrolyzing enzyme is contacted with the oil before the degumming step.
  • suitable degumming methods include water degumming, ALCON oil degumming (e.g., for soybeans), safinco degumming, "super degumming," UF degumming, TOP degumming, uni-degumming, dry degumming and ENZYMAXTM degumming. See e.g. U.S. Patent Nos.
  • water degumming may be performed at elevated temperature, e.g. at 50 to 90°C.
  • the oil/water mixture may be agitated for e.g. 5 to 60 minutes to allow separation of the phosphatides into the water phase, which is then removed from the oil.
  • Acid degumming may also be performed.
  • oil may be contacted with acid (e.g. 0.1 to 0.5% of a 50% solution of citric or malic acid) at 60 to 70°C, mixed, contacted with 1 to 5% water and cooled to 25 to 45 °C.
  • acid e.g. 0.1 to 0.5% of a 50% solution of citric or malic acid
  • WO 2006/008508 Further suitable degumming procedures for use with the process of the present invention are described in WO 2006/008508.
  • the process comprises contacting the chlorophyll- or chlorophyll derivative-hydrolyzing enzyme with the oil and subsequently performing an enzymatic degumming step using an acyltransferase as described in WO 2006/008508.
  • Acyltransferases suitable for use in the process are also described in WO 2004/064537, WO 2004/064987 and WO 2009/024736.
  • acyltransferase activity may be used, particularly enzymes comprising the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues: L, A, V, I, F, Y, H, Q, T, N, M or S.
  • acyltransferase is a mutant Aeromonas salmonicida mature lipid acyltransferase (GCAT) with a mutation of Asn80Asp, e.g. an acyltransferase comprising the amino acid sequence of SEQ ID NO:23 after undergoing post- translational modification (see Figure 23), or an enzyme having at least 80% sequence identity thereto.
  • GCAT Aeromonas salmonicida mature lipid acyltransferase
  • Asn80Asp e.g. an acyltransferase comprising the amino acid sequence of SEQ ID NO:23 after undergoing post- translational modification (see Figure 23), or an enzyme having at least 80% sequence identity thereto.
  • the process comprises a degumming step using a phospholipase.
  • a phospholipase Any enzyme having e.g. a phospholipase Al (E.G.3.1.1.32) or a phospholipase A2 (E.C.3.1.1.4) activity may be used, for example Lecitase Ultra® or pancreatic phospholipase A2 (Novozymes, Denmark).
  • the process comprises contacting the chlorophyll- or chlorophyll derivative-hydrolyzing enzyme with the oil and subsequently performing an enzymatic degumming step using a phospholipase, for example using a degumming step as 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.
  • the degumming step is performed before the chlorophyll or chlorophyll derivative hydrolysis step, preferably the degumming process does not produce lysophospholipids.
  • the degumming step may be a water degumming step.
  • an enzymatic degumming step using an enzyme such as phospholipase C (IUB 3.1.4.1) may be used.
  • Polypeptides having phospholipase C activity which are may be used in a degumming step are disclosed, for example, in WO2008143679, WO2007092314, WO2007055735, WO2006009676 and WO03089620.
  • a suitable phospholipase C for use in the present invention is Purifme®, available from Verenium Corporation, Cambridge, MA.
  • an acid treatment/caustic neutralization step may be performed in order to further reduce phospholipid levels in the oil after water degumming.
  • a single degumming step comprising acid treatment/caustic neutralization may be performed. Such methods are typically referred to as total degumming or alkali refining.
  • an acid treatment/caustic neutralization step is particularly effective in removing products of the enzymatic hydrolysis of chlorophyll, e.g. chlorophyllide, pheophorbide and pyropheophorbide.
  • this step may be performed at any stage in the process after the enzyme treatment step.
  • such a step may comprise addition of an acid such as phosphoric acid followed by neutralization with an alkali such as sodium hydroxide.
  • an acid/caustic neutralization treatment compounds such as chlorophyllide, pheophorbide and pyropheophorbide are extracted from the oil in an aqueous phase.
  • the oil is typically first contacted with 0.05 to 0.5% by weight of concentrated phosphoric acid or citric acid, e.g. at a temperature of 50 to 90°C, and mixed to help precipitate phosphatides.
  • the contact time may be, e.g. 10 seconds to 30 minutes.
  • an aqueous solution of an alkali e.g. 1 to 20% aqueous sodium hydroxide
  • an alkali e.g. 1 to 20% aqueous sodium hydroxide
  • the oil may then be heated to about 90°C and the aqueous soap phase separated from the oil by centrifugation.
  • further wash steps with e.g. sodium hydroxide or water may also be performed.
  • the method of the present invention may optionally involve a step of removing phytol-free derivatives of chlorophyll such as chlorophyllide, pheophorbide and pyropheophorbide.
  • chlorophyll such as chlorophyllide, pheophorbide and pyropheophorbide.
  • Such products may be present in the composition due to the hydrolysis of chlorophyll or a chlorophyll derivative by the enzyme of the invention, or may be present naturally, as a contaminant, or as an undesired component in a processed product.
  • Pyropheophorbide may also be present in the composition due to the breakdown of pheophorbide, which may itself be produced by the activity of an enzyme having pheophytinase activity on pheophytin, or pheophorbide may be formed from chlorophyllide following the action of chlorophyllase on chlorophyll (see Figure 1). Processing conditions used in oil refining, in particular heat, may favour the formation of pyropheophorbide as a dominant component, for instance by favouring the conversion of pheophytin to pyropheophytin, which is subsequently hydrolysed to pyropheophorbide.
  • the process of the present invention reduces the level of chlorophyllide, pheophorbide and/or pyropheophorbide in the oil, compared to either or both of the levels before and after enzyme treatment.
  • the chlorophyllide, pheophorbide and/or pyropheophorbide concentration may increase after enzyme treatment.
  • the process involves a step of removing chlorophyllide, pheophorbide and/or pyropheophorbide such that the concentration of such products is lower than after enzyme treatment.
  • the chlorophyllide, pheophorbide and/or pyropheophorbide produced by this enzymatic step is removed from the oil, such that the final level of these products in the oil is lower than before enzyme treatment.
  • the process may reduce the concentration of chlorophyllide, pheophorbide and/or pyropheophorbide by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%, compared to the concentration of chlorophyllide, pheophorbide and/or pyropheophorbide (by weight) present in the oil before the chlorophyllide, pheophorbide and/or pyropheophorbide removal step, i.e. before or after enzyme treatment.
  • the chlorophyllide, pheophorbide and/or pyropheophorbide concentration in the oil after the removal step may be less than 100, less than 50, less than 30, less than 10, less than 5, less than 1, less than 0.5, less than 0.1 mg/kg, or less than 0.02 mg/kg, based on the total weight of the composition (e.g a vegetable oil).
  • reaction products such as chlorophyllide, pheophorbide and/or pyropheophorbide may be simply and easily removed from the oil by a step such as acid treatment/caustic neutralization.
  • chlorophyll and chlorophyll derivatives may be substantially removed from the oil without the need for further processing steps such as clay and/or silica treatment and deodorization (as indicated by the dashed boxes shown in Fig. 26).
  • the process does not comprise a clay treatment step. Avoiding the use of clay is advantageous for the reasons described earlier, in particular the reduction in cost, the reduced losses of oil through adherence to the clay and the increased retention of useful compounds such as carotenoids and tocopherol.
  • the process may be performed with no clay treatment step and no deodorization step, which results in an increased concentration of such useful compounds in the refined oil, compared to a process involving clay treatment.
  • the process may comprise a step of silica treatment, preferably subsequent to the enzyme treatment.
  • the method may comprise use of an adsorbent-free or reduced adsorbent silica refining devices and processes, which are known in the art, e.g., using TriSyl Silica Refining Processes (Grace Davison, Columbia, MD), or, SORBSIL RTM silicas (INEOS Silicas, Joliet, IL).
  • the silica treatment step may be used to remove any remaining chlorophyllide, pheophorbide and/or pyropheophorbide or other polar components in the oil.
  • a silica treatment step may be used as an alternative to an acid treatment/caustic neutralization (total degumming or alkali refining) step.
  • the process comprises a two-stage silica treatment, e.g. comprising two silica treatment steps separated by a separation step in which the silica is removed, e.g. a filtration step.
  • the silica treatment may be performed at elevated temperature, e.g. at 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.
  • the process may comprise a deodorization step, typically as the final refining step in the process.
  • deodorization refers to steam distillation of the oil, which typically removes volatile odor and flavor compounds, tocopherol, sterols, stands, carotenoids and other nutrients.
  • the oil is heated to 220 to 260°C under low pressure (e.g. 0.1 to 1 kPa) to exclude air.
  • Steam e.g. 1-3% by weight
  • the aqueous distillate may be collected.
  • deodorization may be performed using an inert gas (e.g. nitrogen) instead of steam.
  • the deodoriztion step may comprise bubble refining or sparging with an inert gas (e.g. nitrogen), for example as described by A. V. Tsiadi et al. in "Nitrogen bubble refining of sunflower oil in shallow pools", Journal of the American Oil Chemists' Society (2001), Volume 78 (4), pages 381-385.
  • the gaseous phase which has passed through the oil may be collected and optionally condensed, and/or volatile compounds extracted therefrom into an aqueous phase.
  • the process of the present invention is performed with no clay treatment but comprising a deodorization step.
  • Useful compounds e.g. carotenoids, sterols, stanols and tocopherol
  • a distillate e.g. an aqueous or nitrogenous distillate
  • This distillate provides a valuable source of compounds such as carotenoids and tocopherol, which may be at least partially lost by entrainment in a process comprising clay treatment.
  • Carotenoids may be removed f om the oil during deodorization in both clay-treated and non-clay-treated oil. Typically the removal of coloured carotenoids is controlled in order to produce an oil having a predetermined colour within a specified range of values.
  • the level of carotenoids and other volatile compounds in the refined oil can be varied by modifying the deodorization step. For instance, in an embodiment where it is desired to retain a higher concentration of carotenoids in the oil, the deodorization step may be performed at a lower temperature (e.g. using steam at 200°C or below). In such embodiments it is particularly preferable to avoid a clay treatment step, since this will result in a higher concentration of carotenoids in the refined oil.
  • the processes of the invention further comprise use of lipid acyltransf erases, phospholipases, proteases, phosphatases, phytases, xylanases, amylases (e.g. a-amylases), glucanases, polygalacturonases, galactolipases, cellulases, hemicellulases, pectinases and other plant cell wall degrading enzymes, as well as mixed enzyme preparations and cell lysates.
  • amylases e.g. a-amylases
  • the processes of the invention can be practiced in conjunction with other processes, e.g., enzymatic treatments, e.g., with carbohydrases, including cellulase, hemicellulase and other side degrading activities, or, chemical processes, e.g., hexane extraction of soybean oil.
  • enzymatic treatments e.g., with carbohydrases, including cellulase, hemicellulase and other side degrading activities
  • chemical processes e.g., hexane extraction of soybean oil.
  • the method of the present invention can be practiced in combination with a method as defined in WO 2006031699.
  • a nucleotide sequence (SEQ ID No. 3) encoding a wheat chiorophyliase (SEQ. ID No. 2, hereinafter wheat chlase) was expressed in Bacillus subtilis with the signal peptide of a B. subtilis alkaline protease (aprE) (see Fig. 17).
  • aprE B. subtilis alkaline protease
  • TRI CHL codon optimized gene construct
  • the construct TRI CHL contains 20 nucleotides with a BssHII restriction site upstream to the wheat chlase coding region to allow fusion to the aprE signal sequence and a Pad restriction site following the coding region for cloning into the bacillus expression vector pBNppt.
  • TRI CHL was digested with BssHII and Pad and li gated with T4 DNA ligase into BssHII and Pad digested pBNppt.
  • the ligation mixture was transformed into E. coli TOP 10 cells.
  • the sequence of the BssHII and Pac insert containing the TRI CHL gene was confirmed by DNA sequencing (DNA Technology A/S, Risskov, Denmark) and one of the correct plasmid clones was designated pBN-TRI_CHL ( Figure 18).
  • pBN-TRI_CHL was transformed into B.subtilis strain BG 6002 a derivative of AK 2200, as described in WO 2003/099843.
  • neomycin resistant (neoR) transformant was selected and used for expression of the wheat chlase.
  • the construct CHL_CHL contains 20 nucleotides with a BssHII restriction site upstream to the chlamy chlase coding region to allow fusion to the aprE signal sequence and a Pad restriction site following the coding region for cloning into the bacillus expression vector pBNppt.
  • CHL CHL was digested with BssHII and Pad and ligated with T4 DNA ligase into BssHII and Pad digested pBNppt.
  • the ligation mixture was transformed into E. coli TOP 10 cells.
  • the sequence of the BssHII and Pac insert containing the CHL_CHL gene was confirmed by DNA sequencing (DNA Technology A/S, Risskov, Denmark) and one of the correct plasmid clones was designated pBN-CHL CHL ( Figure 20).
  • pBN-CHL_CHL was transformed into B.subtilis strain BG 6002 a derivative of AK 2200, as described in WO 2003/099843.
  • neomycin resistant (neoR) transformant was selected and used for expression of the chlamy chlase.
  • chlorophyllase from Triticum aestivum was investigated in a crude extracted rapeseed oil (no. 8 from AAK, Sweden) in a low water environment.
  • Crude rapeseed oil was scaled in a Wheaton glass and heated with magnetic stirring to 60°C.
  • Water, enzyme chlororophyllase from Triticum, liquid (54,19 U/ml) or freeze dried powder (41 Units/ml) and NaOH (samples 7 and 12) was added.
  • the sample was treated with high shear mixing for 20 seconds and incubated at 60°C with magnetic stirring.
  • Tables 2 and 3 indicate a clear activity of Triticum chlorophyllase on both pheophytin and pyropheophytin in a low water environment.
  • the enzyme activity was dependent on the amount of water in the reaction mixture.
  • chlorophyllase from Triticum aestivum was investigated in water degummed soya oil (no. 35 from ADM, Hamburg, Germany) in a low water environment.
  • the water degumming process was conducted with 2% water or with 2% water containing a lipid acyltransferase (LysoMax Oil® from Danisco A/S).
  • LysoMax Oil® is an Aeromonas salmonicida mature lipid acyltransferase (GCAT) with a mutation of Asn80Asp, comprising the amino acid sequence of SEQ ID NO:23 (see Fig. 23). This enzyme is known to be very active on phospholipids during formation of lysophospholipids.
  • Lipid acyltransferase (LysoMax Oil®) 100 U/ml ml 0 0.2
  • Lipid acyltransferase activity may be determined as described in WO 2004/064987.
  • the crude soya oil was heated to 55°C. Water and (in sample B) the lipid acyltransferase enzyme was added and the sample was mixed with a high shear mixer for 30 seconds, followed by incubation at 55°C with magnetic stirring. After 30 minutes the sample was heated to 97°C in a water bath to inactivate the enzyme and the samples were centrifuged at 3000 rcf for 3 minutes at 65 °C, and the oil phase collected.
  • Triticum chlorophyllase was tested in these two water degummed oil samples as described in Table 5 :
  • the oil was scaled in a Wheaton glass and heated to 40°C . Enzyme was added and the headspace was purged with nitrogen before the glass was closed with a lid. In all samples the water content is below 0.5% by weight. The samples were incubated for 7 days at 40°C with very gentle magnetic agitation.
  • rows 1 to 4 relate to water degummed oil A (i.e. degummed without lipid acyltransferase) with varying dosages of chlorophyllase as shown
  • rows 5 to 8 relate to water degummed oil B (i.e. degummed with lipid acyltransferase), with varying dosages of chlorophyllase as shown.
  • the results in Table 6 indicate that there is a clear effect of Triticum chlorophyllase with regard to degradation of pheophytin (see Figure 24) and pyropheophytin (see Figure 25) in degummed oil in a low water environment.
  • chlorophyll derivatives may in general be quantified by HPLC analysis according to the following method.
  • HPLC analysis is performed using a method in general terms as described in "Determination of chlorophylls and carotenoids by high-performance liquid chromatography during olive lactic fermentation", Journal of Chromatography, 585, 1991, 259-266.
  • the determination of pheophytin, pheophorbide, pyropheophytin and pyropheophorbide is performed by HPLC coupled to a diode array detector.
  • the column employed in the method is packed with CI 8 material and the chlorophylls were separated by gradient elution. Peaks are assigned using standards of chlorophyll A and B from SigmaAldrich, e.g. based on the representative HPLC chromatogram from Journal of Chromatography, 585, 1991, 259-266 shown in Figure 27.
  • Triticum chlorophyllase was tested in crude rapeseed oil with addition of low amount of water at 60°C and the activity as a function of water concentration and reaction time was followed by HPLC/MS analysis of pheophytin and pyropheophytin. The results clearly showed increased enzyme activity by increasing the water content from 0.111 to 0.5% water. Above 0.5% water the enzyme activity did not further increase. At water concentrations below 0.5% it was possible to run the enzyme reaction without phase separation.
  • chlorophyllase treatment of crude oil can be conducted with a high degree of pheophytin hydrolysis (>90%) within 4 hours reaction time at 60°C. Under these conditions the reaction can take place without phase separation of a gum phase.
  • Triticum chlorophyllase showed high conversion of both pheophytin and pyropheophytin over an extended reaction time (7 days at 40°C).
  • chlorophyllase enzymes may require phospholipids in order to be active on chlorophyll in oil in a high water (two-phase) system. Therefore chlorophyllase treatment of oil may be conducted during or before a water degumming process, at a water content of e.g. 1 to 2%, when phospholipids are still available in the oil. In such a process, the chlorophyllase treatment may require a reaction time of 2 to 4 hours. A long enzyme reaction time of oil in a reactor with 1 to 2% water is however not always preferable because it might be a bottle neck in the oil refining process. Extended treatment of oil with water might also increase the risk of fatty acid formation by oil hydrolysis and loss of product.
  • the activity of chlorophyllase in plant oils in a two phase (high water) system may be dependent on the amount of phospholipids found in the crude oil. Lower amounts of phospholipid may result in reduced activity in such a two-phase system.
  • oils exported as crude oil will always undergo a water degumming before they are shipped overseas from the country of production. Therefore if the chlorophyllase is used in a two-phase (high water) system, the low level of phospholipids remaining in the oil may significantly inhibit the effectiveness of a chlorophyllase treatment performed after export of the oil.
  • the results above surprisingly demonstrate that at a low water content, chlorophyllase is active both in a low and high phospholipid environment.
  • the enzyme may be added to the degummed oil before shipment to overseas destinations.
  • the enzyme is thereby provided with a long incubation time resulting in a high degree of conversion of substrate without delaying the overall production process.
  • phospholipids plays a synergistic effect in bringing the chlorophyll to the boundary of the oil/water interface, thus facilitating the contact between the chlorophyllase enzyme in the water phase and the chlorophyll in the oil phase.
  • the effect of phospholipid on chlorophyllase activity is not so important.

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Abstract

Selon un aspect, cette invention concerne un procédé de traitement d'une huile végétale, ledit procédé comprenant une étape consistant à mettre l'huile en contact avec une enzyme qui est capable d'hydrolyser la chlorophylle ou un dérivé de chlorophylle, l'enzyme étant mise en contact avec l'huile en présence de moins de 0,5 % en poids d'eau.
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Citations (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US5264367A (en) 1991-05-16 1993-11-23 Rohm Gmbh Enzymatic treatment of edible oils
EP0583265A1 (fr) 1991-04-16 1994-02-23 Evotec BioSystems GmbH Procede de preparations de nouveaux biopolymeres
EP0622446A2 (fr) 1993-04-25 1994-11-02 Showa Sangyo Co., Ltd. Procédé de raffinage d'huile et de graisse
US5558781A (en) 1993-11-19 1996-09-24 Metallgesellschaft Aktiengesellschaft Process for enzymatically degumming vegetable oil
EP0752008A1 (fr) 1994-02-17 1997-01-08 Affymax Technologies N.V. Mutagenese d'adn par fragmentation aleatoire et reassemblage
WO1998018912A1 (fr) 1996-10-31 1998-05-07 Novo Nordisk A/S Nouvelle phospholipase, sa production et son utilisation
WO1998031790A1 (fr) 1997-01-16 1998-07-23 Röhm Gmbh Proteine a activite de phospholipase
EP0866796A1 (fr) 1995-09-22 1998-09-30 Medical Research Council Ameliorations relative a la mutagenese d'acides nucleiques
US6001640A (en) 1995-07-26 1999-12-14 Roehm Gmbh Vegetable oil enzymatic degumming process by means of aspergillus phospholipase
WO2000032758A1 (fr) 1998-11-27 2000-06-08 Novozymes A/S Variants d'enzyme lipolytique
US6103505A (en) 1996-12-09 2000-08-15 Novo Nordisk A/S Method for reducing phosphorus content of edible oils
WO2000058517A1 (fr) 1999-03-26 2000-10-05 Diversa Corporation Reassemblage d'acide nucleique en evolution dirigee induit par l'exonuclease
US6162623A (en) 1995-06-27 2000-12-19 Lipton, Division Of Conopco, Inc. Processes for preparing and using immobilized lipases
US6180406B1 (en) 1994-02-17 2001-01-30 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
WO2001027251A1 (fr) 1999-10-14 2001-04-19 Novozymes A/S Lysophospholipase issue de aspergillus
WO2001034835A2 (fr) 1999-11-09 2001-05-17 Max-Planck Gesellschaft zur Förderung der Wissenschaften e.V. Procede de production de biopolymeres a proprietes modifiees
WO2002006457A2 (fr) 2000-07-13 2002-01-24 Maxygen, Inc. Genes de lipase
US6344328B1 (en) 1995-12-07 2002-02-05 Diversa Corporation Method for screening for enzyme activity
US6355693B1 (en) 1995-09-22 2002-03-12 Scotia Lipidteknik Ab Fractionated vegetable oil
US6361974B1 (en) 1995-12-07 2002-03-26 Diversa Corporation Exonuclease-mediated nucleic acid reassembly in directed evolution
WO2002029022A2 (fr) 2000-10-05 2002-04-11 E. I. Du Pont De Nemours And Company Chlorophyllases
US6376689B1 (en) 1999-09-02 2002-04-23 Cargill, Incorporated Removal of gum and chlorophyll-type compounds from vegetable oils
WO2003089620A2 (fr) 2002-04-19 2003-10-30 Diversa Corporation Phospholipases, acides nucleiques codant pour ces phosphalipases et methodes de fabrication et d'utilisation
WO2003099843A2 (fr) 2002-05-20 2003-12-04 Dow Corning Corporation Derives de peptides et utilisation de ces derives pour la synthese de materiaux composites a base de silicium
WO2003100044A1 (fr) 2002-05-29 2003-12-04 Scandinavian Biotechnology Research (Scanbi) Ab Nouvelle acyltransferase amelioree
WO2004064537A2 (fr) 2003-01-17 2004-08-05 Danisco A/S Procede
WO2006008508A1 (fr) 2004-07-16 2006-01-26 Danisco A/S Procede de demucilagination enzymatique
WO2006009676A2 (fr) 2004-06-16 2006-01-26 Diversa Corporation Compositions et procedes pour la decoloration enzymatique de la chlorophylle
WO2006031699A2 (fr) 2004-09-10 2006-03-23 Diversa Corporation Compositions et procedes pour la fabrication et la modification d'huiles
WO2007055735A2 (fr) 2005-05-31 2007-05-18 Verenium Corporation Hydrolases, acides nucleiques codant pour celles-ci et procedes de fabrication et d'utilisation de celles-ci
WO2007092314A2 (fr) 2006-02-02 2007-08-16 Verenium Corporation Estérases, acides nucléiques apparentés et procédés associés
WO2008040465A1 (fr) 2006-10-02 2008-04-10 Ab Enzymes Gmbh Clonage, expression et utilisation de lysophospholipases acides
WO2008143679A2 (fr) 2006-06-01 2008-11-27 Verenium Corporation Acides nucléiques et protéines et procédés pour leur fabrication et leur utilisation
WO2009024736A1 (fr) 2007-08-17 2009-02-26 Danisco A/S Protéine

Patent Citations (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202B1 (fr) 1985-03-28 1990-11-27 Cetus Corp
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
EP0583265A1 (fr) 1991-04-16 1994-02-23 Evotec BioSystems GmbH Procede de preparations de nouveaux biopolymeres
US5264367A (en) 1991-05-16 1993-11-23 Rohm Gmbh Enzymatic treatment of edible oils
EP0622446A2 (fr) 1993-04-25 1994-11-02 Showa Sangyo Co., Ltd. Procédé de raffinage d'huile et de graisse
US5558781A (en) 1993-11-19 1996-09-24 Metallgesellschaft Aktiengesellschaft Process for enzymatically degumming vegetable oil
EP0752008A1 (fr) 1994-02-17 1997-01-08 Affymax Technologies N.V. Mutagenese d'adn par fragmentation aleatoire et reassemblage
US6180406B1 (en) 1994-02-17 2001-01-30 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6162623A (en) 1995-06-27 2000-12-19 Lipton, Division Of Conopco, Inc. Processes for preparing and using immobilized lipases
US6001640A (en) 1995-07-26 1999-12-14 Roehm Gmbh Vegetable oil enzymatic degumming process by means of aspergillus phospholipase
US6355693B1 (en) 1995-09-22 2002-03-12 Scotia Lipidteknik Ab Fractionated vegetable oil
EP0866796A1 (fr) 1995-09-22 1998-09-30 Medical Research Council Ameliorations relative a la mutagenese d'acides nucleiques
EP1103606A2 (fr) 1995-11-30 2001-05-30 Maxygen, Inc. Procédé d'élaboration de polynucléotides présentant des caractéristiques désirées par sélection itérative et recombinaison
EP1138763A2 (fr) 1995-11-30 2001-10-04 Maxygen, Inc. Procédé d'élaboration de polynucléotides présentant des caractéristiques désirées par sélection itérative et recombinaison
US6344328B1 (en) 1995-12-07 2002-02-05 Diversa Corporation Method for screening for enzyme activity
US6361974B1 (en) 1995-12-07 2002-03-26 Diversa Corporation Exonuclease-mediated nucleic acid reassembly in directed evolution
WO1998018912A1 (fr) 1996-10-31 1998-05-07 Novo Nordisk A/S Nouvelle phospholipase, sa production et son utilisation
US6103505A (en) 1996-12-09 2000-08-15 Novo Nordisk A/S Method for reducing phosphorus content of edible oils
WO1998031790A1 (fr) 1997-01-16 1998-07-23 Röhm Gmbh Proteine a activite de phospholipase
WO2000032758A1 (fr) 1998-11-27 2000-06-08 Novozymes A/S Variants d'enzyme lipolytique
WO2000058517A1 (fr) 1999-03-26 2000-10-05 Diversa Corporation Reassemblage d'acide nucleique en evolution dirigee induit par l'exonuclease
US6376689B1 (en) 1999-09-02 2002-04-23 Cargill, Incorporated Removal of gum and chlorophyll-type compounds from vegetable oils
WO2001027251A1 (fr) 1999-10-14 2001-04-19 Novozymes A/S Lysophospholipase issue de aspergillus
WO2001034835A2 (fr) 1999-11-09 2001-05-17 Max-Planck Gesellschaft zur Förderung der Wissenschaften e.V. Procede de production de biopolymeres a proprietes modifiees
WO2002006457A2 (fr) 2000-07-13 2002-01-24 Maxygen, Inc. Genes de lipase
WO2002029022A2 (fr) 2000-10-05 2002-04-11 E. I. Du Pont De Nemours And Company Chlorophyllases
WO2003089620A2 (fr) 2002-04-19 2003-10-30 Diversa Corporation Phospholipases, acides nucleiques codant pour ces phosphalipases et methodes de fabrication et d'utilisation
WO2003099843A2 (fr) 2002-05-20 2003-12-04 Dow Corning Corporation Derives de peptides et utilisation de ces derives pour la synthese de materiaux composites a base de silicium
WO2003100044A1 (fr) 2002-05-29 2003-12-04 Scandinavian Biotechnology Research (Scanbi) Ab Nouvelle acyltransferase amelioree
WO2004064987A2 (fr) 2003-01-17 2004-08-05 Danisco A/S Procede
WO2004064537A2 (fr) 2003-01-17 2004-08-05 Danisco A/S Procede
WO2006009676A2 (fr) 2004-06-16 2006-01-26 Diversa Corporation Compositions et procedes pour la decoloration enzymatique de la chlorophylle
WO2006008508A1 (fr) 2004-07-16 2006-01-26 Danisco A/S Procede de demucilagination enzymatique
WO2006031699A2 (fr) 2004-09-10 2006-03-23 Diversa Corporation Compositions et procedes pour la fabrication et la modification d'huiles
WO2007055735A2 (fr) 2005-05-31 2007-05-18 Verenium Corporation Hydrolases, acides nucleiques codant pour celles-ci et procedes de fabrication et d'utilisation de celles-ci
WO2007092314A2 (fr) 2006-02-02 2007-08-16 Verenium Corporation Estérases, acides nucléiques apparentés et procédés associés
WO2008143679A2 (fr) 2006-06-01 2008-11-27 Verenium Corporation Acides nucléiques et protéines et procédés pour leur fabrication et leur utilisation
WO2008040465A1 (fr) 2006-10-02 2008-04-10 Ab Enzymes Gmbh Clonage, expression et utilisation de lysophospholipases acides
WO2009024736A1 (fr) 2007-08-17 2009-02-26 Danisco A/S Protéine

Non-Patent Citations (43)

* Cited by examiner, † Cited by third party
Title
"Analysis for Phosphorus in Oil by Inductively Coupled Plasma Optical Emission Spectroscopy", AOCS OFFICIAL METHOD CA, 2009, pages 20 - 99
"Bailey's Industrial Oil and Fat Products", 2005, JOHN WILEY & SONS
"Bailey's Industrial Oil and Fat Products", vol. 2.2, 2005, JOHN WILEY & SONS
"Behaviour of Chlorophyll Derivatives in Canola Oil Processing", JAOCS, September 1993 (1993-09-01), pages 837 - 841
"Determination of chlorophylls and carotenoids by high-performance liquid chromatography during olive lactic fermentation", JOURNAL OF CHROMATOGRAPHY, vol. 585, 1991, pages 259 - 266
"Phospholipids in Lecithin Concentrates by Thin-Layer Chromatography", JOURNAL OF CHROMATOGRAPHY A, vol. 864, 1999, pages 179 - 182
"Phospholipids in Vegetable Oils Nephelometric Method", AOCS RECOMMENDED PRACTICE CA, 2009, pages 19 - 86
"Proc. World Conf. Oilseed Edible Oils Process", vol. 1, 1998, AOCS PRESS, pages: 130 - 134
A. V. TSIADI ET AL.: "Nitrogen bubble refining of sunflower oil in shallow pools", JOURNAL OF THE AMERICAN OIL CHEMISTS' SOCIETY, vol. 78, no. 4, 2001, pages 381 - 385, XP002540082, DOI: doi:10.1007/s11746-001-0272-5
ALI KHAMESSAN ET AL., JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, vol. 60, no. 1, 1994, pages 73 - 81
ALTSCHUL ET AL., J. MOL. BIOL., 1990, pages 403 - 410
AOCS RECOMMENDED PRACTICE JA, 2009, pages 7 - 86
ARKUS ET AL: "Mechanistic analysis of wheat chlorophyllase", ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, vol. 438, 2007, pages 146 - 155, XP027195522 *
ARRIAGADA-STRODTHOFF ET AL: "Optimization of chlorophyllase-catalyzed hydrolysis of chlorophyll in monophasic organic solvent media", APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, vol. 142, 2007, pages 263 - 275, XP002678365 *
AUSUBEL ET AL.: "Short Protocols in Molecular Biology", 1999
BEUCAGE S.L. ET AL., TETRAHEDRON LETTERS, vol. 22, 1981, pages 1859 - 1869
BITAR ET AL: "Chlorophyllase biocatalysis in an aqueous/miscible organic solvent medium containing canola oil", JOURNAL OF THE AMERICAN OIL CHEMISTS' SOCIETY, vol. 81, 2004, pages 927 - 932, XP002543901 *
BOCKISCH, M.: "Fats and Oils Handbook, The extraction of Vegetable Oils", 1998, AOCS PRESS, pages: 45 - 445
CARUTHERS MH ET AL., NUC ACIDS RES SYMP SER, 1980, pages 215 - 23
CLAUSEN: "Enzymatic oil degumming by a novel microbial phospholipase", EUR. J. LIPID SCI. TECHNOL., vol. 103, 2001, pages 333 - 340
FEMS MICROBIOL LETT, vol. 174, no. 2, 1999, pages 247 - 50
FEMS MICROBIOL LETT, vol. 177, no. 1, 1999, pages 187 - 8
HIGGINS DG; SHARP PM, GENE, vol. 73, no. 1, 1988, pages 237 - 244
HORN T ET AL., NUC ACIDS RES SYMP SER, 1980, pages 225 - 232
HORNERO-MENDEZ ET AL., FOOD RESEARCH INTERNATIONAL, vol. 38, no. 8-9, 2005, pages 1067 - 1072
HORWELL DC, TRENDS BIOTECHNOL., vol. 13, no. 4, 1995, pages 132 - 134
J. AMER. OIL. CHEM. SOC., vol. 58, 1981, pages 561
J. BIOL. CHEM., vol. 236, 1961, pages 2544 - 2547
JOURNAL OF AMERICAL OIL CHEMISTS' SOCIETY, vol. 83, no. 11, 2006, pages 973 - 979
JOURNAL OF CHROMATOGRAPHY, vol. 585, 1991, pages 259 - 266
KHAMESSAN ET AL: "Biocatalysis of chlorophyllase from Phaeodactylum tricornutum in organic solvent media", PROCESS BIOCHEMISTRY, vol. 30, 1995, pages 159 - 168, XP002678625 *
KIANI ET AL., ANALYTICAL BIOCHEMISTRY, vol. 353, 2006, pages 93 - 98
KLEIN; VISHNIAC, J. BIOL. CHEM., vol. 236, 1961, pages 2544 - 2547
MARCHLER-BAUER, NUCLEIC ACIDS RES., vol. 31, 2003, pages 383 - 387
MASUDA ET AL., EUR. J. BIOCHEM., vol. 202, 1991, pages 783 - 787
MATTHES ET AL., EMBO J., vol. 3, 1984, pages 801 - 805
MORINAGA ET AL., BIOTECHNOLOGY, vol. 2, 1984, pages 646 - 649
N.N.: "102nd AOCS Annual Meeting: Technical Program-Analytical", 102ND AOCS ANNUAL MEETING & EXPO; CINCINNATI, U.S.A; MAY 1-4, 2011, 2011, pages 1 - 16, XP002678366, Retrieved from the Internet <URL:http://www.aocs.org/archives/am2011/abstracts.cfm?interest=Analytical> [retrieved on 20120621] *
NELSON; LONG, ANALYTICAL BIOCHEMISTRY, vol. 180, 1989, pages 147 - 151
SAIKI R K ET AL., SCIENCE, vol. 239, 1988, pages 487 - 491
SCHELBERT ET AL., THE PLANT CELL, vol. 21, 2009, pages 767 - 785
SIMON RJ ET AL., PNAS, vol. 89, no. 20, 1992, pages 9367 - 9371
YI, J. OF MOLECULAR CATALYSIS B: ENZYMATIC, vol. 19, 2002, pages 319 - 325

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