FIELD

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.

BACKGROUND

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.

For example, vegetable oils derived from oilseeds such as soybean, palm or rape seed (canola), cotton seed and peanut oil typically contain some chlorophyll. However the presence of high levels of chlorophyll pigments in vegetable oils is generally undesirable. This is because chlorophyll imparts an undesirable green colour and can induce oxidation of oil during storage, leading to a deterioration of the oil.

Various methods have been employed in order to remove chlorophyll from vegetable oils. Chlorophyll may be removed during many stages of the oil production process, including the seed crushing, oil extraction, degumming, caustic treatment and bleaching steps. However the bleaching step is usually the most significant for reducing chlorophyll residues to an acceptable level. During bleaching the oil is heated and passed through an adsorbent to remove chlorophyll and other colour-bearing compounds that impact the appearance and/or stability of the finished oil. The adsorbent used in the bleaching step is typically clay.

In the edible oil processing industry, the use of such steps typically reduces chlorophyll levels in processed oil to between 0.02 to 0.05 ppm. However 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. Also 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. Thus attempts have been made to remove chlorophyll from oil by other means, for instance using the enzyme 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. Typically 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). 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. 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.

Depending on the processing conditions, 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.

It has been proposed to treat plant oils with a chlorophyllase in the presence of water, for instance during a water degumming process (e.g. at a water content of 1 to 2% by weight, based on the total weight of oil). However, such a process may require a step of incubation with the enzyme in the presence of water lasting several hours. This step may therefore cause an inconvenient delay in the overall process, and/or the modification of specific physical components of the process (e.g. expansion of the capacity of degumming reactors). Moreover, extended treatment of oil with water may increase the risk of fatty acid formation by oil hydrolysis and consequent loss of product.

There is a still a need for an improved process for removing chlorophyll and chlorophyll derivatives such as pheophytin and pyropheophytin from plant oils. In particular, there is a need for a more convenient process, which is fast and efficient and requires minimal modification of existing production facilities. SUMMARY

In one aspect 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. hi one embodiment, 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.

In one embodiment, the plant oil is a crude plant oil. For instance, the enzyme may be contacted with a crude oil before a step of degumming of the oil. In an alternative embodiment, the plant oil is a degummed plant oil (e.g. an oil which has been treated to remove phophatides/phospholipids). For instance, 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). Preferably the degumming step comprises water degumming.

In some embodiments, 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.

In further embodiments, 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. For instance, in some embodiments 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.

In further embodiments, 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. For instance, in some embodiments 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. In one specific embodiment, 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. In this embodiment, the oil is preferably a water-degummed oil. This step may take place, for example, during transport or shipping of the oil.

In an alternative embodiment, the enzyme is contacted with the oil at a temperature of 55°C to 65°C for 1 to 4 hours. In this embodiment, the oil is preferably a crude plant oil.

In one embodiment the enzyme comprises a chlorophyllase, pheophytinase, pyropheophytinase or pheophytin pheophorbide hydrolase. For example, the enzyme may comprise a polypeptide sequence as defined in any one of SEQ ID NOs: 1, 2, 4, 6 or 8 to 15, or a functional fragment or variant thereof. Preferably the enzyme comprises a polypeptide sequence 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. In particularly preferred embodiments, 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.

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

As described herein, it has surprisingly been demonstrated that chlorophyllases are active in a one phase system comprising up to 0.5% by weight water. Accordingly, in the present invention 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. Advantageously, according to the present invention 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. Moreover, due to the extended incubation time which may be provided by utilising, for example, the transport or shipping phase of a degummed oil, the enzyme may typically be used at a low dose, thereby reducing cost. In view of the expected differences between one and two phase systems, e.g. in terms of differing enzyme stability, access to substrate and co-factors, the present demonstration of chlorophyllase activity in a one phase system is a surprising result.

BRIEF DESCRIPTION OF DRAWINGS

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).

Figure 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.

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.

Figure 27 shows an HPLC chromatogram using absorbance detection (430 nm) indicating numbered peaks associated with: 1 = chlorophyllide b; 2 = chlorophyllide a; 3 = neoxanthin; 3' = neoxanthin isomer; 4 = neochrome; 5 = violaxanthin; 6 = luteoxanthin; 7 = auroxanthin; 8 = anteraxanthin; 8' = anteraxanthin isomer; 9 = mutatoxanthin; 10 = lutein; 10' = lutein isomer; 10" = lutein isomer; 11 = pheophorbide b; 12 = pheophorbide a; 13 = chlorophyll b; 13' - chlorophyll b'; 14 = chlorophyll a; 14' = chlorophyll a'; 15 = pheopytin b; 15' = pheophytin b'; 16 = β-carotene; 17 = pheophytin a; 17' = pheophytin a'; 18 = pyropheophytin b; 19 = pyropheophytin a.

DETAILED DESCRIPTION

In one aspect the present invention relates to a process for treating a plant oil. Typically the process is used to remove chlorophyll and/or chlorophyll derivatives from the oil, or to reduce the level of chlorophyll and/or chlorophyll derivatives in the oil, for instance where the chlorophyll and/or chlorophyll derivatives are present as a contaminant.

Chlorophyll and chlorophyll derivatives

By "chlorophyll 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. Thus the presence of chlorophyll and chlorophyll derivatives in an oil, can give such an oil an undesirable green, greenish or brownish colour. In one embodiment, 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. These compounds still contain the colour-bearing porphyrin ring, with chlorophyllide being green and pheophorbide and pyropheophorbide a reddish brown colour. In some embodiments, it may also be desirable to remove these phytol-free derivatives and to reduce the green/red/brown colouring in the oil. Thus in one embodiment of the invention, the process may further comprise a step of removing or reducing the level of phytol-free chlorophyll derivatives in the oil.

The chlorophyll or chlorophyll derivative may be either a or b forms. Thus as used herein, the term "chlorophyll" 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.

Plant oils

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.

In preferred embodiments, 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). Examples of 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).

Chlorophyll and chlorophyll derivatives in oil

The chlorophyll and/or chlorophyll derivatives (e.g. chlorophyll, pheophytin and/or pyropheophytin) may be present in the oil naturally, as a contaminant, or as an undesired component in a processed product. The chlorophyll and/or chlorophyll derivatives (e.g. chlorophyll, pheophytin and/or pyropheophytin) may be present at any level in the oil. Typically chlorophyll, pheophytin and/or pyropheophytin may be present as a natural contaminant in the oil at a concentration of 0.001 to 1000 mg/kg (0.001 to 1000 ppm, 10 ^"7 to 10 ^"1 wt %), based on the total weight of the oil. In further embodiments, 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.

Phytol-free chlorophyll derivatives may also be present in the oil. For instance, chlorophyllide, pyropheophorbide and/or pyropheophorbide may be present at any level in the oil. Typically 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. In further embodiments, 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.

Enzymes hydrolysing chlorophyll or a chlorophyll derivative

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. Typically "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. Thus the enzyme typically has an esterase or hydrolase activity. Preferably the enzyme has esterase or hydrolase activity in an oil phase, and optionally also in an aqueous phase.

Thus the enzyme may, for example, be a chlorophyllase, pheophytinase or pyropheophytinase. Preferably, the enzyme is capable of hydrolysing at least one, at least two or all three of chlorophyll, pheophytin and pyropheophytin. In a particularly preferred embodiment, the enzyme has chlorophyllase, pheophytinase and pyropheophytinase activity. In further embodiments, two or more enzymes may be used in the method, each enzyme having a different substrate specificity. For instance, 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. By "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. For example, hydrolytic activity may be detected using fluorescence-based techniques. In one suitable assay, a polypeptide to be tested for hydrolytic activity on chlorophyll or a chlorophyll derivative is incubated in the presence of a substrate, and product or substrate levels are monitored by fluorescence measurement. Suitable substrates include e.g. chlorophyll, pheophytin and/or pyropheophytin. Products which may be detected include chlorophyllide, pheophorbide, pyropheophorbide and/or phytol.

Assay methods for detecting hydrolysis of chlorophyll or a chlorophyll derivative are disclosed in, for example, Ali Khamessan et al. (1994), Journal of Chemical Technology & Biotechnology, 60(1), pages 73 - 81 ; Klein and Vishniac (1961), J. Biol. Chem. 236: 2544-2547; and Kiani et al. (2006), Analytical Biochemistry 353: 93-98. Alternatively, 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. In one embodiment, 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:

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

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.

In preferred embodiments, 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.

In a further embodiment, hydrolytic activity on chlorophyll or a chlorophyll derivative may be determined using a method as described in EP10159327.5.

Chlorophyllases

In one embodiment, 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. For example, 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) can be used in the process.

In one embodiment 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. In one aspect, the chlorophyllase may be an enzyme as described in WO 0229022 or WO 2006009676. For example, the Arabidopsis thaliana chlorophyllase can be used as described, e.g. in NCBI entry NM_123753. Thus the chlorophyllase may be a polypeptide comprising the sequence of SEQ ID NO:l (see Figure 2). In another embodiment, the chlorophyllase is derived from algae, e.g. from Phaeodactylum tricornutum.

In another embodiment, the chlorophyllase is derived from wheat, e.g. from Triticum sp., especially from Triticum aestivum. For example, 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).

In another embodiment, the chlorophyllase is derived from Chlamydomonas sp., especially from Chlamydomonas reinhardtii. For example, 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).

Pheophytin pheophorbide hydrolase

In one embodiment, the enzyme is capable of hydrolyzing pheophytin and pyropheophytin. For example, 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. However PPH is inactive on chlorophyll. As described in Schelbert et al., PPH orthologs are commonly present in eukaryotic photosynthesizing organisms. PPHs represent a defined sub-group of α/β hydrolases which are phylogenetically distinct from chlorophyllases, the two groups being distinguished in terms of sequence homology and substrates.

In specific embodiments of the invention, 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. For example, in one embodiment, 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.

In further embodiments, 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. For example, 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:

Table 1

Organism Accession Genbank ID

Arabidopsis thaliana NP 196884 15240707

Populus trichocarpa XP 002314066 224106163

Vitis vinifera CAO40741 157350650

Oryza sativa (japonica) NP 001057593 115467988

Zea mays ACF87407 194706646

Nicotiana tabacum CA099125 156763846

Ostreococcus lucimarinus XP 001415589 145340970

Ostreococcus tauri CAL50341 116000661

Physcomitrella patens XP 001761725 168018382

Phaeodactylum tricornutum XP 002181821 219122997

Chlamydomonas reinhardtii XP 001702982 159490010

Micromonas sp. RCC299 ACO62405 226516410

For example, 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 Functional variants and fragments of known sequences which hydrolyse chlorophyll or a chlorophyll derivative may also be employed in the present invention. By "functionar' it is meant that the fragment or variant retains a detectable hydrolytic activity on chlorophyll or a chlorophyll derivative. Typically such variants and fragments show homology to a known chlorophyllase, pheophytinase or pyropheophytinase sequence, e.g. at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a known chlorophyllase, pheophytinase or pyropheophytinase amino acid sequence, e.g. to SEQ ID NO:l or any one of SEQ ID NOs: 1, 2, 4, 6 or 8 to 15, e.g. over a region of at least about 10, 20, 30, 50, 100, 200, 300, 500, or 1000 or more residues, or over the entire length of the sequence.

The percentage of sequence identity may be determined by analysis with a sequence comparison algorithm or by a visual inspection. In one aspect, the sequence comparison algorithm is a BLAST algorithm, e.g., a BLAST version 2.2.2 algorithm.

Other 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. For example, 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). In some embodiments, 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.

Isolation and production of enzymes

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.

For example, 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.

Alternatively, 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.

In a yet further alternative, 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. In 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). The term "nucleotide sequence" as used herein 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.

Typically, the nucleotide sequence encoding a polypeptide having chlorophyllase, pheophytinase and/or pyropheophytinase activity is prepared using recombinant DNA techniques. However, in an alternative embodiment of the invention, 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).

Modification of enzyme sequences

Once 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).

Instead of site directed mutagenesis, such as described above, one can introduce mutations randomly for instance using a commercial kit such as the GeneMorph PCR mutagenesis kit from Stratagene, or the Diversify PCR random mutagenesis kit from Clontech. 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.

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

As a non-limiting example, in addition, 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.

The application of the above-mentioned and similar molecular evolution methods allows the identification and selection of variants of the enzymes of the present invention which have preferred characteristics without any prior knowledge of protein structure or function, and allows the production of non-predictable but beneficial mutations or variants. There are numerous examples of the application of molecular evolution in the art for the optimisation or alteration of enzyme activity, such examples include, but are not limited to one or more of the following: optimised expression and/or activity in a host cell or in vitro, increased enzymatic activity, altered substrate and/or product specificity, increased or decreased enzymatic or structural stability, altered enzymatic activity/specificity in preferred environmental conditions, e.g. temperature, H, substrate.

As will be apparent to a person skilled in the art, using molecular evolution tools an enzyme may be altered to improve the functionality of the enzyme. Suitably, a nucleotide sequence encoding an enzyme (e.g. a chlorophyllase, pheophytinase and/or pyropheophytinase) used in the invention may encode a variant enzyme, i.e. 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.

Polypeptide sequences

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.

As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "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 μg of the freeze-dried material may be dissolved in 50 μΐ 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 μΐ of 45 mM dithiothreitol. After cooling to room temperature, 5 μΐ 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.

135 μΐ of water and 5 μg of endoproteinase Lys-C in 5 μΐ of water may be added to the above reaction mixture and the digestion may be carried out at 37°C under nitrogen for 24 hours. The resulting peptides may be separated by reverse phase HPLC on a VYDAC C18 column (0.46χ15αη;10μιη; The Separation Group, California, USA) using solvent A: 0.1% TFA in water and solvent B: 0.1% TFA in acetonitrile. Selected peptides may be re-chromatographed on a Develosil CI 8 column using the same solvent system, prior to N-terminal sequencing. Sequencing may be done using an Applied Biosystems 476A sequencer using pulsed liquid fast cycles according to the manufacturer's instructions (Applied Biosystems, California, USA).

Sequence comparison

Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, 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.

In the present context, 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. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although 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.

In the present context, 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). Typically, the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence. Although 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.

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.

However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the Vector NTI Advance™ 11 (Invitrogen Corp.). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al 1999 Short Protocols in Molecular Biology, 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 Advance™ 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.).

Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, 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 Advance™ 11 package.

Alternatively, percentage homologies may be calculated using the multiple alignment feature in Vector NTI Advance™ 11 (Invitrogen Corp.), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244). Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

Should Gap Penalties be used when determining sequence identity, then preferably the default parameters for the programme are used for pairwise alignment. For example, the following parameters are the current default parameters for pairwise alignment for BLAST 2: FOR BLAST2 DNA PROTEIN

EXPECT 10 10

THRESHOLD

WORD SIZE 11 3

SCORING PARAMETERS

Match/Mismatch 2, -3 n/a

Scores

Matrix n/a BLOSUM62

Gap Costs Existence: 5 Existence: 11 Extension: 2 Extension: 1

In one embodiment, preferably the 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.

For the purposes of the present invention, 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 Advance™ 11 (Invitrogen Corp.). For pairwise alignment the scoring parameters used are preferably BLOSUM62 with Gap existence penalty of 1 land Gap extension penalty of 1.

Suitably, 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. Suitably, the degree of identity with regard to a nucleotide sequence may be determined over the whole sequence.

Amino acid mutations

The 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. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

ALIPHATIC Non-polar G A P

I L V

Polar - uncharged C S T M

N Q

Polar - charged D E

K R

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. from one class of residue to another or alternatively involving the inclusion of 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 β- 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. For the avoidance of doubt, "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. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al, PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

Nucleotide sequences

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. For the purposes of the present invention, it is to be understood that 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. In addition, 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.

Alternatively, such 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. 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.

In general, 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. For example, enzymes used in the methods of the invention can be formulated to be amphipathic or more lipophilic. For example, 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. In one aspect, 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. In various aspects of the invention, 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. For example, 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 μιηοΐ 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.

In a preferred embodiment, 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.

Crude and degummed oils

In one embodiment, the enzyme is contacted with a crude plant oil. In an alternative embodiment, the enzyme is contacted with a degummed oil. Thus 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. Following an enzymatic degumming step (e.g. using a phospholipase) or a total degumming step (e.g. comprising an acid treatment/caustic neutralization) the phospholipid content 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:

Canola Rapeseed Soybean

Crude oil <2.5 <3.5 <4.0

Water-degummed oil <0.6 <0.8 <0.4

Acid-degummed oil <0.1 <0.2 The values in the table above are taken from Bailey's Industrial Oil and Fat Products til

(2005), edition, Ed. by Fereidoon Shahidi, John Wiley & Sons, and the phospholipid content of other oils is also described therein or is well-known in the art. The phospholipid content of oils 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. 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".

Thus in one embodiment, the crude oil is an oil comprising at least 0.5%, at least 1.0% or at least 2% by weight phospholipid. In another embodiment, the oil is a water degummed plant oil comprising 0.1 to 1% by weight phospholipid.

Lysophospholipid content

In a preferred embodiment of the process, the enzyme is contacted with the oil at a time when a concentration of lysophospholipid in the oil is as low as possible. For instance, the enzyme may be contacted with the oil in the presence of less than 0.2% by weight lysophosholipid. By "in the presence of less than 0.2% by weight 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. In some embodiments where the process comprises an enzymatic degumming step using an enzyme which generates lysophospholipids, the enzyme which hydrolyses chlorophyll or a chlorophyll derivative may be contacted with the oil before the enzymatic degumming step. Alternatively 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.

In one embodiment, 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.

In particular embodiments, the lysophospholipid content of the oil is less than 0.2%, less than 0.15%, less than 0.1% or less than 0.05% by weight, based on the total weight of 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. as described above for phospholipids, including using HPLC or TLC analysis methods. Suitable methods are described in AOCS Recommended Practice Ja 7-86 (reapproved 2009), "Phospholipids in Lecithin Concentrates by Thin-Layer Chromatography" or Journal of Chromatography A, 864 (1999) 179-182.

Temperature

In general 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.

At higher temperatures pheophytin is decomposed to pyropheophytin, which is generally less preferred because some chlorophyllases are less active on pyropheophytin compared to pheophytin. In addition, 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.

In view of the above, in particularly preferred embodiments the oil is incubated with the enzyme at below about 80°C, preferably below about 70°C, preferably at about 68°C or below, preferably at about 65 °C or below, in order to reduce the amount of conversion to pyropheophytin. However, in embodiments where the reaction time is relatively short (e.g. less than 24 hours, typically less than about 4 hours), in order to keep a good reaction rate it is preferred to keep the temperature of the oil above 50 °C during incubation with the enzyme. Accordingly 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.

In alternative embodiments, for instance where the enzyme is contacted with the oil during transport or shipping of the oil, a lower temperature may be used (typically in combination with a longer reaction time). In these embodiments 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. For instance, in one embodiment the enzyme may be contacted with the oil at ambient temperature, e.g. 15 to 25°C.

Preferably 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.

Reaction time

Suitably 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. For example, the reaction time may be at least about 1 minute, more preferable at least about 5 minutes, more preferably at least about 10 minutes. In some embodiments 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. In some embodiments, the reaction time may up to 6 hours, or up to 24 hours.

In alternative embodiments, e.g. using a lower reaction temperature as described above, the reaction temperature may be extended, for instance to include the duration of a transport or shipping step of the oil. In specific embodiments, 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.

Water content

In embodiments of the present invention, 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. For instance the 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. Preferably the water content is at least 0.1% by weight, more preferably at least 0.2%, 0.3% or 0.4% by weight. Thus 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.

Typically the enzyme is contacted with the oil in 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

Preferably the process is carried out between about pH 4.0 and about pH 10.0, more preferably between about pH 5.0 and about pH 10.0, more preferably between about pH 6.0 and about pH 10.0, more preferably between about pH 5.0 and about pH 7.0, more preferably between about pH 5.0 and about pH 7.0, more preferably between about pH 6.5 and about pH 7.0, e.g. at about pH 7.0 (i.e. neutral pH). In one embodiment preferably the process is carried out between about pH 5.5 and pH 6.0.

Oil separation

Following an enzymatic treatment step using an enzyme according to the present invention, in one embodiment the treated liquid (e.g. oil) is separated with an appropriate means such as a centrifugal separator and the processed oil is obtained. Upon completion of the enzyme treatment, if necessary, 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 The process of the present invention involving an enzyme treatment typically reduces the level of chlorophyll and/or chlorophyll derivatives in the oil. For example, 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. Thus in particular embodiments, the concentration of chlorophyll and/or chlorophyll derivatives in the oil after treatment may be less than 100, less than 50, less than 30, less than 10, less than 5, less than 1, less than 0.5, less than 0.1 mg kg or less than 0.02 mg/kg, based on the total weight of the oil.

Further processing steps

In a typical plant oil processing method, 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.

In embodiments of the present invention, 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. In particular embodiments the enzyme is preferably contacted with the oil before the degumming step. In another embodiment, 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). In some embodiments, 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. Thus 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.

Degumming

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. Thus as used herein, the term "degumming" means the refining of oil by removing phospholipids from the oil. In some embodiments, 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. Thus 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 ENZYMAX™ degumming. See e.g. U.S. Patent Nos. 6,355,693; 6,162,623; 6,103,505; 6,001,640; 5,558,781; 5,264,367, 5,558,781; 5,288,619; 5,264,367; 6,001,640; 6,376,689; WO 0229022; WO 98118912; and the like. Various degumming procedures incorporated by the methods of the invention are described in Bockisch, M. (1998), Fats and Oils Handbook, The extraction of Vegetable Oils (Chapter 5), 345-445, AOCS Press, Champaign, Illinois. Water degumming typically refers to a step in which the oil is incubated with water (e.g. 1 to 5% by weight) in order to remove phosphatides. Typically 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. For example, 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.

Further suitable degumming procedures for use with the process of the present invention are described in WO 2006/008508. In one embodiment 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. Any enzyme having acyltransferase activity (generally classified as E.G.2.3.1) 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. In one embodiment, 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.

In another embodiment, the process comprises a degumming step using 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). In one embodiment 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. In embodiments where the degumming step is performed before the chlorophyll or chlorophyll derivative hydrolysis step, preferably the degumming process does not produce lysophospholipids. For example, in these embodiments the degumming step may be a water degumming step. In another such embodiment, an enzymatic degumming step using an enzyme such as phospholipase C (IUB 3.1.4.1) may be used. Polypeptides 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.

Acid treatment/caustic neutralization

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

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

In such methods, 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. Subsequently an aqueous solution of an alkali (e.g. 1 to 20% aqueous sodium hydroxide) is added, e.g. at a temperature of 50 to 90°C, followed by incubation and mixing for 10 seconds to 30 minutes. The oil may then be heated to about 90°C and the aqueous soap phase separated from the oil by centrifugation. Optionally, further wash steps with e.g. sodium hydroxide or water may also be performed.

Chlorophyllide, pheophorbide and pyropheophorbide removal

Thus the method of the present invention may optionally involve a step of removing phytol-free derivatives of chlorophyll such as chlorophyllide, pheophorbide and pyropheophorbide. Such products may be present in the composition due to the hydrolysis of chlorophyll or a chlorophyll derivative by the enzyme of the invention, or may be present naturally, as a contaminant, or as an 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.

In one embodiment 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. Thus in some embodiments the chlorophyllide, pheophorbide and/or pyropheophorbide concentration may increase after enzyme treatment. Typically 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. Preferably 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.

For example, 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. Thus in particular embodiments, 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).

It is an advantage of the present process that 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. Thus in preferred embodiments 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).

Clay treatment

It is particularly preferred that 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.

In some embodiments, 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.

Silica treatment

Although not always required, in some embodiments the process may comprise a step of silica treatment, preferably subsequent to the enzyme treatment. For example, 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 R™ 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. For example, in some embodiments a silica treatment step may be used as an alternative to an acid treatment/caustic neutralization (total degumming or alkali refining) step.

In one embodiment 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.

Deodorization

In some embodiments, the process may comprise a deodorization step, typically as the final refining step in the process. In one embodiment, deodorization refers to steam distillation of the oil, which typically removes volatile odor and flavor compounds, tocopherol, sterols, stands, carotenoids and other nutrients. Typically the oil is heated to 220 to 260°C under low pressure (e.g. 0.1 to 1 kPa) to exclude air. Steam (e.g. 1-3% by weight) is blown through the oil to remove volatile compounds, for example for 15 to 120 minutes. The aqueous distillate may be collected.

In another embodiment, deodorization may be performed using an inert gas (e.g. nitrogen) instead of steam. Thus 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.

In some embodiments, 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) may be at least partially extracted from the oil in a distillate (e.g. an aqueous or nitrogenous distillate) obtained from the deodorization step. 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.

The loss of tocopherol during bleaching depends on bleaching conditions and the type of clay applied, but 20-40% removal of tocopherol in the bleaching step has been reported (K. Boki, M, Kubo, T. Wada, and T. Tamura, ibid., 69, 323 (1992)). During processing of soy bean oil a loss of 13% tocopherol in the bleaching step has been reported (S. Ramamurthi, A. R. McCurdy, and R. T. Tyler, in S. S. Koseoglu, K. C. Rhee, and R. F. Wilson, eds., Proc. World Conf. Oilseed Edible Oils Process, vol. 1 , AOCS Press, Champaign, Illinois, 1998, pp. 130-134).

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.

Further enzyme treatments

In further aspects, 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. In alternative aspects, 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. In one embodiment the method of the present invention can be practiced in combination with a method as defined in WO 2006031699. The invention will now be further illustrated with reference to the following non- limiting examples.

EXAMPLE 1

Cloning and expression of a chiorophyliase from Triticum aestiviim (wheat) in

Bacillus subtilis

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). For optimal expression in Bacillus, a codon optimized gene construct (TRI CHL) was ordered at GenScript (GenScript Corporation, Piscataway, NJ 08854, USA).

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.

The construct 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.

One neomycin resistant (neoR) transformant was selected and used for expression of the wheat chlase.

EXAMPLE 2

Cloning and expression of a chiorophyliase from Chlamydomonas reinhardtii (green algae) in Bacillus subtilis A nucleotide sequence (SEQ ID No. 5) encoding a Chlamydomonas chloryphyllase (SEQ. ID No. 4, hereinafter chlamy chlase) was expressed in Bacillus subtilis with the signal peptide of a B. subtilis alkaline protease (aprE) (see Fig.s 19 and 20). For optimal expression in Bacillus, a codon optimized gene construct (CHL_CHL) was ordered at GenScript (GenScript Corporation, Piscataway, NJ 08854, USA).

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.

The construct 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.

One neomycin resistant (neoR) transformant was selected and used for expression of the chlamy chlase.

EXAMPLE 3

Chlorophyllase treatment of crude rapeseed oil in a low water environment

The activity of chlorophyllase from Triticum aestivum (see Example 1) was investigated in a crude extracted rapeseed oil (no. 8 from AAK, Sweden) in a low water environment.

12 samples were prepared as described in Table 1 : Table 1. Chlorophyllase treatment of oil in low water environment

Crude rapeseed oil was scaled in a Wheaton glass and heated with magnetic stirring to 60°C. Water, enzyme (chlorophyllase 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.

Samples were taken out after 2 and 4 hours reaction time. The samples were centnfuged and analysed by HPLC/MS with results shown in Table 2. Based on the HPLC/MS analysis, the degree of hydrolysis of pheophytin and pyropheophytin was calculated with results shown in Table 3.

- 46 -

Table 2. HPLC/MS analysis of pheophytins and pheophorbides

Sample pg/g= ppm pg/g= ppm pg/g= ppm pg/g= ppm pg/g= ppm Ratio a/a' : no. Hour Pheophorbide_a Pheoph lin_a Pheophytin_a' Pheophtyin a+a' Pyropheophytin_a a/fa+ayiOOVo

1 2 0.31 1.63 0.64 2.27 0.25 71.65

2 2 0.56 1.15 0.46 1.61 0.19 71.51

3 2 1.08 0.57 0.26 0.83 0.15 68.29

4 2 1.70 0.16 0.1 1 0.27 0.10 58.60

5 2 1.50 0.09 0.08 0.17 0.08 51.60

6 2 1.59 0.08 0.08 0.16 0.08 49.96

7 2 0.76 0.11 0.04 0.16 0.1 1 71.36

8 2 0.40 1.45 0.56 2.01 0.23 72.17

9 2 1.51 0.91 0.36 1.27 0.19 71.72

10 2 2.28 0.38 0.19 0.57 0.14 67.15

11 2 2.63 0.12 0.09 0.22 0.10 56.74

12 2 0.74 0.03 0.02 0.05 0.09 62.28

Sample pg/g= ppm pg/g= ppm pg/g= ppm pg/g= ppm pg/g= ppm Ratio a/a' : no. Hour Pheophorbide_a Pheophytin_a Pheophylin_a' Pheophtyin a+a' Pyropheophytin_a a/(a+a')* 00%

1 4 0.44 1.61 0.59 2.20 0.24 73.02

2 4 0.70 0.97 0.41 1.39 0.17 70.35

3 4 1.23 0.35 0.17 0.52 0.12 66.67

4 4 1.54 0.06 0.06 0.12 0.07 52.84

5 4 1.65 0.03 0.04 0.07 0.05 44.54

6 4 1.71 0.03 0.04 0.07 0.05 41.85

7 4 0.77 0.02 0.02 0.04 0.07 57.69

8 4 0.65 1.41 0.51 1.92 0.20 73.40

9 4 2.02 0.61 0.25 0.86 0.14 71.13

10 4 2.72 0.19 0.10 0.30 0.10 64.67

11 4 1.82 0.03 0.04 0.08 0.06 43.69

12 4 0.75 0.005 0.01 0.01 0.05 31.56

Table 3. Degree of hydrolysis of pheophytin and pyropheophytin

Sample Degree of hydrolysis Degree of hydrolysis

no. Hour Pheophtyin a+a' Pyropheophytin_a

1 2 0 0

2 2 29 25

3 2 63 42

4 2 88 59

5 2 93 67

6 2 93 67

7 2 93 55

8 2 12 10

9 2 44 25

10 2 75 45

11 2 90 61

12 2 98 64

Sample

no. Hour Pheophtyin a+a' Pyropheophytin_a

1 4 0 0

2 4 37 31

3 4 76 50

4 4 94 70

5 4 97 80

6 4 97 80

7 4 98 71

8 4 13 17

9 4 61 41

10 4 87 57

11 4 97 76

12 4 99 79

The results in 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.

The result from the test with the freeze dried chlorophyllase (samples 2 to 6) was evaluated statistically using Statgraphic ANOVA. The enzyme response on pheophytin and pyropheophytin is illustrated graphically in Figures 21 and 22. A clear increase in activity on both pheophytin and pyropheophytin is seen by increasing the water content form 0.11 to 0.5%. Above 0.5% water no significant changes in enzyme activity is seen. In the experiments with water content below 0.5% no two phase formation and gum separation was observed. The results thus indicate that it is possible to treat crude oil with Triticum chlorophyllase at water concentrations below 0.5% in a one phase system without gum separation and still obtain a very high degree of hydrolysis. For pheophytin more than 90% was hydrolysed and 70% pyropheophytin was hydrolysed after 4 hours reaction time.

EXAMPLE 4

Chlorophyllase treatment of water degummed soya oil in a low water environment

The activity of chlorophyllase from Triticum aestivum (see Example 1) 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. These two samples of water degummed soya oil reflect two commonly used procedures to produce water degummed oil in the refining industry.

Water degumming was performed using samples A and B as described in Table 4:

Table 4. Recipe for water degumming of soya oil

A B

Crude Soya Oil no.35 g 200 200

Lipid acyltransferase (LysoMax Oil®) 100 U/ml ml 0 0.2

Extra Water ml 4.000 3.800

Lipid acyltransferase U/g oil 0.00 0.10

% water 2.00 2.00

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 :

Table 5. Recipes for chlorophyllase treatment of water degummed soya oil

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.

After 7 days the samples were analysed by HPLC/MS with results shown in Table 6:

Table 6

* = missing value

In Table 6 above, rows 1 to 4 relate to water degummed oil A (i.e. degummed without lipid acyltransferase) with varying dosages of chlorophyllase as shown, and 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. At a chlorophyllase dosage of 0.1 U/g oil, pheophytin and pyropheophytin are almost completely hydrolysed in the water degummed (WDG) oil without use of a lipid acyltransferase.

In the WDG oil treated with a lipid acyltransferase, more chlorophyllase enzyme is needed to completely degrade pyropheophytin. The results indicate that the reaction product of the lipid acyltransferase (lysophospholipids) in the water degummed oil may have an inl ibitory effect on chlorophyllase degradation of pyropheophytin. However, it is possible to compensate for the inhibitory effect by adding more chlorophyllase.

HPLC analysis

In the examples described herein, 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.

Conclusion

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.

It is concluded that 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. h addition, in a one phase system with chlorophyllase added to a degummed oil with total amount of water below 0.5%, it was demonstrated that Triticum chlorophyllase showed high conversion of both pheophytin and pyropheophytin over an extended reaction time (7 days at 40°C). The results also indicated that the chlorophyllase activity in water degummed oil treated with a lipid acyltransferase was reduced compared with chlorophyllase activity in water degummed oil produced without using the lipid acyltransferase. The reduced activity of chlorophyllase in water degummed oil produced with a lipid acyltransferase could however be compensated by increasing the enzyme dosage.

It has previously been shown that it is possible to remove the chlorophyll components by treating vegetable oil with a chlorophyllase. This enzyme facilitates the hydrolysis of phytol from the chlorophyll components during formation of chlorophyllide, pheophorbide or pyropheophorbide, which under weak alkaline condition are water soluble and can be washed out of the oil.

However, 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.

In contrast, the results above demonstrate that it is possible to perform the chlorophyllase treatment of crude oil before the water degumming process, by adding the enzyme to the oil with a small amount of water such that a one phase system remains. By running the chlorophyllase treatment of oil in a one phase system, it is possible to conduct this process in storage and buffer tanks without the need to expand tank capacity of the degumming reactors. Another advantage of a one phase system is that diffusion of the chlorophyll substrate is faster. In addition enzymes are typically more thermostable at low water levels.

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. Typically 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. hi contrast, the results above surprisingly demonstrate that at a low water content, chlorophyllase is active both in a low and high phospholipid environment. This provides the opportunity to perform the chlorophyllase treatment in a degummed oil. For instance, 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. Without being bound by theory, it is expected that in a two phase (high water) system, 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. In a low water environment, however, it appears that the effect of phospholipid on chlorophyllase activity is not so important.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.