WO2013160374A1 - Process for refining crude plant oil involving enzymatic hydrolysis and gum recycling - Google Patents

Abstract

In one aspect, provided herein is a continuous process for refining a crude plant oil, comprising (i) an enzymatic hydrolysis step comprising contacting the crude oil with an enzyme which hydrolyses chlorophyll or a chlorophyll derivative; and (ii) a degumming step comprising separating a gum phase from the oil; wherein the process comprises recycling at least a portion of the separated gum phase for addition to the crude oil before enzymatic hydrolysis.

Description

PROCESS FOR REFINING CRUDE PLANT OIL INVOLVING ENZYMATIC HYDROLYSIS AND GUI- RECYCLING.

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 32). 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 hydro lyzing 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 32). 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 32). Pyropheophorbide is less polar than pheophorbide resulting in the pyropheophoribe 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.

WO2011/1 10967 discloses that the activity of chlorophyllases in plant oils may be dependent on the phospholipid content, and that lysophospho lipids have a negative impact on chlorophyllase activity. This document therefore suggests contacting chlorophyllases with crude plant oils (e.g. before degumming or other refining steps), because crude oils typically contain relatively high levels of phospholipids and low levels of lysophospholipids.

However, chlorophyllase activity in some crude plant oils may still be sub-optimal under certain circumstances. Therefore 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 process in which chlorophyll and chlorophyll derivatives are removed with enhanced efficiency from crude plant oils.

SUMMARY

In one aspect the present invention provides a continuous process for refining a crude plant oil, comprising (i) an enzymatic hydrolysis step comprising contacting the crude oil with an enzyme which hydro lyses chlorophyll or a chlorophyll derivative; and (ii) a degumming step comprising separating a gum phase from the oil; wherein the process comprises recycling at least a portion of the separated gum phase for addition to the crude oil before enzymatic hydrolysis.

In one embodiment, the crude oil comprises less than 1% by weight, preferably less than 0.5% by weight, phospholipid before addition of the gum phase.

In one embodiment, the crude oil comprises at least 1% by weight, preferably at least 1.5% by weight, phospholipid after addition of the gum phase.

In one embodiment, the crude oil is obtained by pressing oil- containing plant seeds. For instance, the crude oil may comprise crude rapeseed (canola) oil, e.g. obtained by pressing of rapeseed.

In one embodiment, the enzyme is contacted with the oil in the presence of 1 to 5% by weight water. Preferably the degumming step comprises water degumming.

In one embodiment, the gum phase is separated from the oil by centrifugation. Preferably the process does not comprise a step of clay treatment.

The enzyme may comprise, for example, a chlorophyllase, pheophytinase, pyropheophytinase or pheophytin pheophorbide hydrolase. In specific embodiments, the enzyme comprises a polypeptide sequence as defined in any one of SEQ ID NOs: 1 to 31, 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 to 31 over at least 50 amino acid residues.

In a further aspect, the present invention provides a refined plant oil obtainable by a process as defined in any preceding claim.

It has surprisingly been found that in embodiments of the present invention, the activity of chlorophyllases in crude oils having a relatively low phospholipid content can be improved by recycling the gum phase back into the crude oil in a continuous refining process. In particular, gum recycling increases the phospholipid content of the oil during the enzymatic treatment step, thereby improving the reaction kinetics and favouring rapid hydrolysis of chlorophyll and chlorophyll derivatives to phytol-free compounds. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows the amino acid sequence of an Arabidopsis thaliana chlorophyllase (SEQ ID NO: l).

Figure 2 shows the amino acid sequence of an Arabidopsis thaliana chlorophyllase (SEQ ID NO:2).

Figure 3 shows the amino acid sequence of Citrus sinensis chlorophyllase (SEQ ID NO:3).

Figure 4 shows the amino acid sequence of a Triticum aestivum chlorophyllase (SEQ ID NO:4).

Figure 5 shows the amino acid sequence of a Triticum aestivum chlorophyllase (SEQ ID NO:5).

Figure 6 shows the amino acid sequence of a Brassica oleracea chlorophyllase (SEQ ID NO:6).

Figure 7 shows the amino acid sequence of a Brassica oleracea chlorophyllase (SEQ ID NO:7).

Figure 8 shows the amino acid sequence of a Brassica oleracea chlorophyllase (SEQ ID NO:8).

Figure 9 shows the amino acid sequence of a Zea Mays chlorophyllase (SEQ ID NO: 9).

Figure 10 shows the amino acid sequence of a Zea Mays chlorophyllase (SEQ ID NO: 10).

Figure 11 shows the amino acid sequence of a Phyllostachys edulis chlorophyllase (SEQ ID NO: l l).

Figure 12 shows the amino acid sequence of a Chenopodium album chlorophyllase (SEQ ID NO: 12).

Figure 13 shows the amino acid sequence of a Ricinus communis chlorophyllase (SEQ ID NO: 13).

Figure 14 shows the amino acid sequence of a Glycine max chlorophyllase (SEQ ID NO: 14). Figure 15 shows the amino acid sequence of a Ginkgo biloba chlorophyllase (SEQ ID NO: 15).

Figure 16 shows the amino acid sequence of a Pachira macrocarpa chlorophyllase (SEQ ID NO: 16).

Figure 17 shows the amino acid sequence of a Populus trichocarpa chlorophyllase (SEQ ID NO: 17).

Figure 18 shows the amino acid sequence of a Sorghum bicolor chlorophyllase (SEQ ID NO: 18).

Figure 19 shows the amino acid sequence of a Sorghum bicolor chlorophyllase (SEQ ID NO: 19).

Figure 20 shows the amino acid sequence of a Vitis vinifera chlorophyllase (SEQ ID NO:20).

Figure 21 shows the amino acid sequence of a Physcomitrella patens chlorophyllase (SEQ ID NO:21).

Figure 22 shows the amino acid sequence of a Aquilegia chlorophyllase (SEQ ID NO:22).

Figure 23 shows the amino acid sequence of a Brachypodium distachyon chlorophyllase (SEQ ID NO:23).

Figure 24 shows the amino acid sequence of a Medicago truncatula chlorophyllase (SEQ ID NO:24).

Figure 25 shows the amino acid sequence of a Piper betle chlorophyllase (SEQ ID NO:25).

Figure 26 shows the amino acid sequence of a Lotus japonicus chlorophyllase (SEQ ID NO:26).

Figure 27 shows the amino acid sequence of a Oryza sativa Indica chlorophyllase (SEQ ID NO:27).

Figure 28 shows the amino acid sequence of a Oryza sativa Japonica chlorophyllase (SEQ ID NO:28). Figure 29 shows the amino acid sequence of a Oryza sativa Japonica chlorophyllase (SEQ ID NO:29).

Figure 30 shows the amino acid sequence of a Picea sitchensis chlorophyllase (SEQ ID NO:30).

Figure 31 shows the amino acid sequence of a Chlamydomonas chlorophyllase (SEQ ID NO:31).

Figure 32 shows the reactions involving chlorophyll and derivatives and enzymes used in the present invention.

Figure 33 shows a diagrammatic representation of a plant oil refining process in one embodiment of the present invention.

Figure 34 shows the results of liquid chromatography mass spectrometry analysis of pheophytin in crude canola oil without and with gum recycling.

Figure 35 shows the results of liquid chromatography mass spectrometry analysis of pyropheophytin in crude canola oil without and with gum recycling.

Figure 36 shows amino acid and nucleotide sequences showing the fusion of a chlorophyllase gene to a His tag and thrombin site.

Figure 37 shows a schematic presentation of an E. coli. expression vector pET28-TRI_CHL containing the TRI CHL gene encoding a chlorophyllase from Triticum aestivum (database acc. no. BT009214).

DETAILED DESCRIPTION

In one aspect the present invention relates to a continuous process for refining a crude 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. By "continuous" it is meant that, in one embodiment, crude oil may be fed continuously into the process, such that at the same time different steps may be performed on oil which has progressed to different stages of the process. In particular, in embodiments of the present invention the gum phase obtained during a degumming step may be added back to crude oil at an earlier stage in the process.

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 hydro lyse 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 process may involve bleaching or de-colorizing to remove the green and/or red/brown colouring of 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.

Chlorophyll and chlorophyll derivatives may exist as a pair of epimers determined by the stereochemistry around the carbon number 13² (numbering according to the IUPAC system). Thus chlorophyll a exists as the pair of epimers chlorophyll a and chlorophyll a ', and chlorophyll b comprises b and b' forms. Pheophytin a comprises the epimers a and a' and pheophytin b comprises b and b' forms. The prime (') forms have S-stereochemistry and non- prime forms have R-stereochemistry about the carbon 13² atom. When used generally herein, the term "chlorophyll and chlorophyll derivatives" includes both prime and non-prime forms.

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 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.

In embodiments of the present invention, the enzyme is contacted with a crude plant oil. According to the present invention, it is preferable to select a crude plant oil having a relatively low phospholipid content, e.g. rapeseed (canola) oil.

Preparation of oil-containing seeds

A crude plant oil may be obtained from various types of oil-containing seeds using known methods. In some embodiments, the seeds may first be subjected to various treatments such as cleaning, conditioning and/or flaking, e.g. as described in Bailey's Industrial Oil and Fat Products (2005), 6^th edition, Ed. by Fereidoon Shahidi, John Wiley & Sons, Chapter 2. Seeds may be flaked, for example, using smooth-surface rolling mills in one or more stages. In an embodiment of a single stage flaking process, flakes of e.g. rapeseed having a thickness of about 0.3 mm may be produced in a single step. In one embodiment of a two-stage method, suitable for use with e.g. rapeseed, a flake thickness of about 0.4-0.7 mm is produced by a first set of rolls, and then flakes of 0.2-0.3 mm thickness are produced in a second stage. Flaking ruptures the cell walls and helps to start the process of releasing oil from the seeds.

Pressing

In one embodiment, the oil is a crude pressed oil, e.g. the oil is obtained by pressing of an oil- containing plant seed. In one embodiment, the oil is crude pressed rapeseed oil, e.g. oil obtained by expeller pressing of rapeseed. By "pressing" it is intended to refer to any application of mechanical force, which typically results in expulsion of a significant proportion of the oil from the oilseeds. This step may be performed using any suitable apparatus known in the art, e.g. continuous screw presses, expellers, single-screw or twin- screw extruders. Pressing may be performed, for example, using a one-stage or multistage process.

Expeller pressing typically reduces the oil content of the seed from e.g. (in the case of rapeseed) about 40% to about 20%. Solvent extraction methods, as are well known in the art, may then be used, if desired, to recover the remaining oil from the press-cake. Oils obtained by pressing (especially pressed rapeseed oil) typically have a relatively low phospholipid content, and are therefore particularly suitable for use in the process of the present invention.

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 "hydro lyzing 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 hydro lytic 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. In 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.

In a further embodiment, enzyme activity may be determined using an assay as described in WO2011/125028. One unit of enzyme activity is defined as the amount of enzyme which hydro lyzes 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.

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 -hydro lyase 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. Typically the chlorophyllase is also capable of hydrolyzing pheophytin and/or pyropheophytin at least to some extent.

In one embodiment the enzyme is a chlorophyllase classified under the Enzyme Nomenclature classification (E.C. 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. In one embodiment, the Arabidopsis thaliana chlorophyllase can be used as described, e.g. in NCBI entry NP 199199. Thus the chlorophyllase may be a polypeptide comprising the sequence of SEQ ID NO:2 (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:4 (see Figure 4). Further suitable chlorophyllase sequences are shown in Table 1 below, and in Figures 1 to 31 (SEQ ID NO:s 1 to 31):

Table 1. Chlorophyllases with accession numbers and names used herein.

Multiple chlorophyllase amino acid sequences show the conserved sequence motif GHSRG (SEQ ID NO: 32). In particular, the serine residue at the active site of the enzyme, which is present in this motif, is highly conserved. In preferred embodiments, the enzyme used in the present invention comprises an enzyme as shown in Table 1 above and/or any of Figures 1 to 31 , comprising any one of SEQ ID NOs 1 to 31, or comprising the sequence motif of SEQ ID NO:32, including fragments, variants and derivatives thereof. These chlorophyllases are typically capable of hydro lysing at least pheophytin (and optionally also pyropheophytin), in addition to chlorophyll.

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 chlorophy liases, 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. In particular, 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 2, or a functional fragment or variant thereof:

Table 2

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

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 "functional" it is meant that the fragment or variant retains a detectable hydro lytic 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 any one of SEQ ID NOs: 1 to 31, 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, many chlorophyllases comprise the conserved sequence motif GHSRG (SEQ ID NO: 32), which as mentioned above comprises a serine residue present at the active site. Conserved sequence motifs found in PPH enzymes include the following: LPGFGVG (SEQ ID NO:33), DFLGQG (SEQ ID NO:34), GNSLGG (SEQ ID NO:35), LVKGVTLLNATPFW (SEQ ID NO:36), HPAA (SEQ ID NO:37), EDPW (SEQ ID NO:38), and SPAGHCPH (SEQ ID NO: 39). In some embodiments, an enzyme for use in the present invention may comprise one or more of these sequences. The GNSLGG (SEQ ID NO:35) motif present in PPH enzymes 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, ligated 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 K 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, EP1138763, EP1103606. 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, pH, 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 CI 8 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 THRESHOLD 10 10

WORD SIZE 11 3

SCORING PARAMETERS

Match/Mismatch Scores 2, -3 n/a

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 phenylglycine. 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 PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive 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 pyropheophytinase 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 μηιοΐ 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. Phospholipid content of the oil before gum recycling

The phospholipid content of plant oils varies according to the particular source and nature of the oil (see Bailey's Industrial Oil and Fat Products (2005), 6^th edition, Ed. by Fereidoon Shahidi, John Wiley & Sons). As well as the plant species from which the oil is derived, environmental factors (such as growing temperature) and the oil extraction technique can affect phospholipid content. For instance, the phospholipid content of some crude plant oils (e.g. soy bean oil) may be up to 5% by weight.

However, the crude oil which is selected for refining according to the method of the present invention typically has a relatively low content of phospholipid. For example, the phospholipid content of the oil at the start of the process (e.g. before addition of the gum phase) may be less than 1% by weight, e.g. based on the total weight of the oil composition.

Typically the phospholipid content of the crude oil is 0.1% by weight or above at the start of the process. The phospholipid is typically present as a natural component of a crude plant oil. Phospholipids commonly found in crude plant oils include phosphatidyl choline (PC), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), phosphatidyl serine (PS) and phosphatidic acid (PA). In preferred embodiments, the phospholipid comprises one or more of PC, PI, PE, PS and PA. Phospholipids are typically present in crude oils in the form of lecithin, the major component of which is PC. Thus in one embodiment, the phospholipid comprises lecithin. The term lecithin as used herein encompasses phosphatidyl choline, phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidyl serine and phosphatidic acid.

In particular embodiments, the phospholipid (e.g. PC, PI, PE, PS, PA and/or lecithin) content of the oil at the start of the process is less than 1.0%, 0.9%, 0.8%, 0.7%, 0.6% or 0.5% by weight, e.g. based on the total weight of the oil. For example, the phospholipid content of the oil may be 0.1 to 1.0%, 0.2 to 0.8%, 0.3 to 0.7%, 0.3 to 0.6% or 0.3 to 0.5% by weight, e.g. based on the total weight of the oil.

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".

Lysophospholipid content

In embodiments of the present invention, it is preferred that the lysophospholipid content of the oil is as low as possible. Preferably the lysophospholipid content in the oil is less than 0.2% by weight, e.g. based on the total weight of the oil composition. The lysophospholipid content in the oil may be any value below 0.2% by weight, including zero.

Lysophospho lipids 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. In preferred embodiments, the concentration of LPC and/or LPE is less than 0.2%>, less than 0.15%, less than 0.1 % or less than 0.05%> by weight, based on the total weight of oil.

The lysophospholipid content of oils may be 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.

Gum recycling

In embodiments of the present invention, at least a portion of a gum phase separated from the oil in the degumming step is recycled back into the crude oil entering the process. By this it is meant that gum is continuously separated from oil which has been treated with the chlorophyllase, and at least a part of the gum phase is re-used to increase the phospholipid content of a subsequent volume of oil entering the process (i.e. before enzymatic hydrolysis of chlorophyll or chlorophyll derivatives takes place). In this manner, the phospholipid content of the oil during the enzymatic treatment can be raised to a level which results in increased chlorophyllase activity. By "gum" or "gum phase" it is meant the predominantly aqueous phase obtained following a degumming step, e.g. the phase separated from the oil following centrifugation of a water- treated oil. Typically the gum comprises predominantly phospholipids, e.g. lecithins such as phosphatidylcholine. For instance, the gum phase may comprise approximately 65% phospholipids and approximately 35% triglycerides.

The present invention involves recycling at least a portion of the gum phase. By this it is meant that part or all of the gum phase may be recycled back into the oil, with or without separation or purification of specific components of the gum phase. In preferred embodiments, at least a portion of a phospholipid component of the gum phase is recycled. The phospholipid component of the gum may correspond to the phospholipid component of the oil as discussed above, and may contain one or more of the individual phospholipids mentioned therein. As used in the present disclosure, the terms "phosphatide" and "phospholipid" may generally be considered to be substantially equivalent in meaning.

For instance, in some embodiments a phospholipid component of the gum may be separated from a triglyceride component of the gum, and only the phospholipid component added back into the oil at the start of the process. Methods for separating phospholipids from oils are known in the art, and are used e.g. in the production of deoiled lecithin. In another embodiment, the gum phase may first be dried before recycling back into the oil, i.e. to separate the dry matter present in the gum phase from water. In one embodiment, the portion of the gum phase which is recycled back into the oil comprises a dried phospholipid component. As used herein, when referring in general to "gum" or a "gum phase" it is therefore intended to encompass such portions or components obtained from the gum phase, especially a phospholipid component thereof.

In further embodiments, the process may further comprise recycling at least a portion of the enzyme (e.g. chlorophyllase) present in the gum phase back into the oil, for example in addition to the phospholipid component. After separation of the gum phase from the oil, the enzyme is expected to be present predominantly in the aqueous gum phase. Thus by recycling the entire gum phase at least a portion of the added enzyme can be reused in the process. Alternatively, the enzyme can be recovered or isolated from the gum phase using known protein purification techniques and added directly back into oil entering the process. In embodiments of the present invention, at least a portion of the gum phase may be recycled back to the crude oil before enzymatic hydrolysis. By this it is meant that the gum is added to oil entering the process before enzymatic hydrolysis has begun or while enzymatic hydrolysis is continuing, e.g. at least before hydrolysis of chlorophyll or chlorophyll derivatives by the chlorophyllase has been completed. Thus the gum phase may be added back into the crude oil before, after, or simultaneously with the addition of the enzyme, provided that the enzyme is still active when the gum phase is added. Preferably the gum phase is added to the crude oil before or at the same time as the enzyme.

The gum phase can be added to the crude oil, preferably with mixing, in any suitable manner. Preferably the gum phase is added back continuously into the crude oil, e.g. such that each volume of oil entering the process receives a particular amount of additional gum. In this way the phospholipid content of the oil during the enzymatic hydrolysis step may be kept at a steady, elevated level, as described below.

Phospholipid content of the oil after gum recycling

The recycling of the gum phase or portion thereof advantageously increases the phospholipid content of the crude oil, thereby increasing chlorophyllase activity during the enzymatic hydrolysis step. Thus the phospholipid content of the oil after addition of the gum phase is preferably at least 1% by weight, e.g. based on the total weight of the oil composition. In particular embodiments, the phospholipid (e.g. PC, PI, PE, PS, PA and/or lecithin) content of the oil after gum recycling is at least 1.1%, 1.2%, 1.3%, 1.4%, 1.5% or 2.0% by weight, e.g. based on the total weight of the oil. For example, the phospholipid content of the oil after gum recycling may be 1.0 to 2.0%, 1.1 to 1.9%, 1.2 to 1.8%, 1.3 to 1.7% or 1.4 to 1.6% by weight, e.g. based on the total weight of the oil. The phospholipid content of the oil after gum addition may be determined using standard methods, as described supra (see e.g. J. Amer. Oil. Chem. Soc. 58, 561 (1981)).

The proportion of the gum phase which is recycled can be varied in order to achieve a desired content of phospholipids during the enzymatic hydrolysis step. For instance, assume that the crude oil comprises 0.3% by weight phospholipid at the start of the process, and it is desired to raise the phospholipid content to 1.2% for the enzymatic reaction step. Once the process has reached a steady-state, recycling approximately 75% of the recovered gum phase (based on the dry mass equivalent of phospholipid) into an equivalent volume of oil entering the process should maintain the required value, assuming that 100% of the phospholipid is recovered in the degumming step. In other words, the process involves adding back 0.9% phospholipid from a volume of oil (out of 1.2% recovered phospholipid in the recovered gum phase) to the 0.3% phospholipid present naturally in an equivalent volume of crude oil entering the process. If the recycling ratio was increased to 80%, the phospholipid content during enzymatic hydrolysis could potentially be increased to 1.5%.

For instance, in one embodiment the gum phase is recycled in a continuous process until the gum concentration in a holding tank has reached a concentration of 1.2%. If oil comprising 0.3%) phospholipid is pumped into the reactor in a continuous process at a rate of 100 tons/hour and all the gum is recycled for 3 hours, the gum concentration in the reactor will be 1.2%. After 3 hours, 25% of the recovered gum may be continuously removed from the process (the remainder being recycled), and the process should maintain a steady state with 1.2% phopspholipid in the reactor.

In practice, the required recycling ratios may need to be modified somewhat due to less than complete recovery of phospholipids during degumming. Thus the recycling ratio may need to be determined empirically, for example by measuring the phospholipid content of the oil before and after addition of gum using one of the methods described above. However, the above illustration is intended merely to indicate how recycling varying proportions of the gum phase may be used to achieve different phospholipid contents during the chlorophyll hydrolysis step.

Enzyme reaction conditions

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 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.

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.

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.

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. In another embodiment, the process is carried out between about pH 6.0 to pH 6.8, e.g. between about pH 6.3 and pH 6.5, preferably about pH Suitably the water content of the oil when incubated (or admixed) with the enzyme is between about 0.5 to about 5% water, more preferably between about 1 to about 3% and more preferably between about 1.5 and about 2% by weight. In specific embodiments, the water content may be, for example, 0.7% to 1.2%, e.g. about 1% by weight; or 1.7% to 2.2%, e.g. about 2% by weight.

When an immobilised enzyme is used, suitably the water activity of the immobilised enzyme may be in the range of about 0.2 to about 0.98, preferably between about 0.4 to about 0.9, more preferably between about 0.6 to about 0.8.

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.

Degumming

In embodiments of the present invention, the process comprises a degumming step. The degumming step in oil refining serves to separate phosphatides (gum or phospholipids) by the addition of water. 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. 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.

Preferably the degumming step comprises water degumming. 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.

In one embodiment, the enzymatic hydrolysis step is performed before the degumming (e.g. water degumming) step. Alternatively, the enzymatic hydrolysis and degumming steps may be performed substantially simultaneously. If the enzyme is added to the oil in a sufficient volume of water, separation of an aqueous gum phase will occur, thereby removing the phospholipids from the oil.

Oil and gum phase separation

Following the degumming step, in one embodiment the gum phase is separated from the oil with an appropriate means such as a centrifugal separator. If necessary, the processed oil can be additionally washed with water or organic or inorganic acid such as, e.g., acetic acid, citric acid, phosphoric acid, succinic acid, and the like, or with salt solutions.

As described above, in the present process at least a portion of the gum phase obtained from the degumming step is recycled back into crude oil entering the process. In some embodiments, a portion of the gum phase (which is not recycled into the process) may be 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 (e.g. 30-50%). Lecithin may be deoiled to reduce the triglyceride content to below 5%.

Further processing steps

The present process may comprise one or more further treatment steps used in a typical plant oil processing method, e.g. acid addition/caustic neutralization, silica treatment, and deodorization. In one embodiment, the process may comprise a series of steps as set out in Figure 33. 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.

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.

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, 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 32). 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 hydro lysed 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 treatment.

Clay treatment

It is particularly preferred that the process reduces the need for clay treatment of the oil. For instance, the process may reduce the amount of clay required by at least 50%>, 70%>, 80%>, 90%), 95%) or 99%) by weight, e.g. compared to the amount of clay required to treat the oil in the absence of a chlorophyllase treatment step. In one embodiment, 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, stanols, carotenoids and other nutrients. Typically the oil is heated to 220 to 260°C under low pressure (e.g. 0.1 to 1 kPa) to exclude air. Steam (e.g. 1-3% 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 from 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 acyltransferases, 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

Effect of gum enrichment on Arabidopsis chlorophyllase activity

To illustrate the effect of gum enrichment of the crude oil, two experiments with chlorophyllase were set up according to recipe 1 and 2, using a chlorophyllase from Arabidopsis thaliana expressed in E. coli.

Cloning in E. coli

Synthetic genes encoding Arabidopsis thaliana (ARA_CHL2, SEQ ID NO:2) and Triticum aestivum (TRI CHL, SEQ ID NO:4, see Example 2 below) chlorophyllases were prepared. Each gene was codon optimized for expression in E. coli. For cloning purposes the genes were extended in the 5 '-end to contain a restriction site for Nhel and in the 3 '-end to contain a restriction site for Xhol.

Following digestion with Nhel and Xhol restriction enzymes the synthetic DNA was ligated into the E. coli expression vector pET-28a(+) (Novagen) digested with the same restriction enzymes. This vector includes a T7 promoter with a Lac operator for controlling expression of inserted genes. The chlorophyllase genes were fused in frame to a His tag and a thrombin cleavage site for purification (example shown in Figure 36). The resulting constructs (an example pET28-TRI_CHL is shown in Figure 37), were transformed into competent E. coli TOP 10 cells (Invitrogen), and plasmids were isolated from transformed colonies and subjected to nucleotide sequencing to verify the correct sequence and that all fusions were as expected.

Expression in E. coli For expression the plasmids were transformed into the expression host E. coli BL21(DE3) (Novagen). The cells were cultured at 37°C in LB containing carbenicillin (50mg/ml) until OD₆oo 0.6-0.8. For induction the culture was added 1 mM IPTG and incubated at 25°C for another 20-24 h before harvesting the cells by centrifugation. The recombinant chlorophyllases were released from the cell pellet by sonication and cellular debris removed by centrifugation.

A) Recipe 1

Crude rape seed oil was heated to 65 °C. Water and enzyme was mixed and added to the oil. The sample was mixed with a high shear mixer (Ultra Turrax) for 30 seconds, followed by incubation with magnetic stirring at 65°C. After 45, 90, 150 and 240 minutes a 1 ml sample was taken out into an Eppendorf tube and centrifuged at 10000 rcf for 10 minutes. The gum phase was separated from the oil and the oil phase was analyzed by liquid chromatography- mass spectrometry (LC-MS) with results shown in Table 1. After 240 min reaction time the remaining sample was centrifuged at 10000 rcf and 65°C for 10 minutes. The oil phase was separated from the gum phase and the gum phase was used in experiment B below (see Recipe 2).

Table 1 : LC-MS analysis of Pheophytin and pyropheophytin in oils

Reaction time Pheophtyin a+a' Pyropheophytin a

hr μg/g= ppm μg/g= ppm

0 8.097 4.299

0.75 4.07 2.67

1.5 1.44 1.17 2.5 0.41 0.44

4 0.09 0.12

B) Recipe 2

Crude canola oil and gum was heated to 65°C during agitation. Water and enzyme was mixed and added to the oil. The sample was mixed with a high shear mixer (Ultra Turrax) for 30 seconds, followed by incubation with magnetic stirring at 65°C. After 30, 60, 120 and 240 minutes 1 ml sample is taken out into an Eppendorf tube and centrifuged at 10000 rcf for 10 minutes. The oil phase was analyzed by LC-MS with results shown in Table 2.

Table 2 LC-MS analysis of pheophytin and pyropheophytin in samples taken from experiment in Table 2

The results from LC-MS analysis of pheophytin and pyropheophytin in Table 1 and 2 are illustrated graphically in Fig.s 34 and 35.

In the experiments above a crude canola oil from Canada was used. This oil had a phosphorus content of 375 ppm corresponding to 0.80% by weight phospholipids. This level of phospholipids is considered low in a crude canola oil, although the level of phospholipids in crude canola oil may vary significantly. The main phospholipid in this oil is phosphatidic acid (average molecular weight = 685).

In experiment B the crude oil was supplemented with 2.5% gum from experiment A. The water content in the gum phase was 70% by weight and thus 0.75%> gum (on dry matter) was added to experiment B. The gum phase from experiment A was checked for residual enzyme activity , and no residual activity was found.

The results from Table 1 and 2 illustrated in Fig. 34 and 35 clearly demonstrate increased activity of the chlorophyllase when the reaction mixture was supplemented with extra phospholipids. If we calculate the rate constant for the degradation of pheophytin in the two experiments (assuming it is a first order enzyme reaction), it was found that the initial rate constant (in the first 1 to 2 hrs) increased by more than 50% when the extra gum phase was added.

Conclusion

The velocity of chlorophyllase reactions in crude vegetable oil to which is added 2 to 3 % by weight water depends on the amount of phospholipids in the gum phase. In certain types of oil the phospholipid content is as low as 0.3-0.5%, which might be a limiting factor for the use of chlorophy liases. It has however been demonstrated above that it is possible to increase chlorophyllase reaction speed by adding phospholipid (gum) to the crude oil. In a continuous oil refining process the gum enrichment can be implemented by recycling part of the gum phase to facilitate the optimum reaction conditions for the chlorophyllase reaction.

In certain types of oils like crude rape seed oil produced by oil pressing, the amount of phospholipids is typically in the range of 0.25 to 0.5%>. When the phospholipid content of the oil is are below about 1%, the chlorophyllase activity may be significant lower. The level of phospholipids in crude oil might therefore limit the utility of chlorophy liases in oil refining. In particular, more enzyme would be needed, or the activity of chlorophyllase might be so low that it is not possible to degrade chlorophyll, pheophytin and pyropheophytin to a level needed for green color removal. The method described herein is capable of overcoming these limitations. It has been demonstrated above that during the oil refining process it is possible to recycle part of the gum phase produced and thus improve the activity of chlorophyllase in a continuous oil refining process. The recycling of the gum phase can take place as illustrated in Fig. 33. By controlling the split ratio of the gum phase it is possible to adjust the crude oil to a level where the activity of the added chlorophyllase is not limited by the amount of phospholipids.

EXAMPLE 2

Effect of gum recycling on Triticum chlorophyllase activity

Crude rape seed oil from AarhusKarlshamn (containing 1.2% phospholipid) was heated to 55°C, added to 1.5% by weight water and treated with high shear mixing for 20 seconds using an Ultra Turrax Mixer. The sample was then incubated for 30 min at 55°C with magnetic agitation. The sample was then transferred to a centrifuge tube and centrifuged at 1870 rcf for 3 minutes. The oil phase was isolated from the gum phase and the gum phase was dried in a rotating vacuum evaporator. The remaining water content in the gum phase was 10.7% by weight after drying.

Triticum aestivum chlorophyllase (TRI CHL, SEQ ID NO:4) was expressed in E. coli as described above in Example 1. The water degummed oil phase and the dried gum phase was recombined and treated with chlorophyllase according the recipe in Table 3 :

Table 3

Sample no. 1 2 3 4 5

Water degummed oil g 10 10 10 10 10

Dried gum phase g 0 0.02 0.05 0.1 0.2 water ml 0.338 0.338 0.338 0.338 0.338

Triticum chlorophyllase (TRI CHL,

SEQ ID NO:4), 12.5 U/ml ml 0.012 0.012 0.012 0.012 0.012

U/g

Chlorophyllase dosage in oil oil 0.015 0.015 0.015 0.015 0.015 Water % 3.50 3.50 3.50 3.50 3.50

Temperature °C 65 65 65 65 65

Water degummed (WDG) oil, containing 0.075% residual phospholipid, and dried gum was scaled in a Wheaton glass and heated to 65°C. Water and enzyme was added. The samples were mixed with a high shear mixer for 20 seconds followed by magnetic stirring at 450 rpm at 65°C for 4 hours. 1 gram sample was taken out after 4 hours and centrifuged at 10000 rcf for 5 minutes. The oil phase was isolated and analysed by LC-MS with results shown in Table 4.

Table 4: LC-MS analysis of pheophytin in oil treated with chlorophyllase

Conclusion

The results from LC-MS analysis of residual pheophytin in the chlorophyllase treated oil confimed that addition of phospholipid (gum) to the water degummed oil improved the activity of the enzyme and contributed to increased degradation of pheophytin in the oil.

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.

Claims

1. A continuous process for refining a crude plant oil, comprising:

(i) an enzymatic hydrolysis step comprising contacting the crude oil with an enzyme which hydrolyses chlorophyll or a chlorophyll derivative; and

(ii) a degumming step comprising separating a gum phase from the oil; wherein the process comprises recycling at least a portion of the separated gum phase for addition to the crude oil before enzymatic hydrolysis.

2. A process according to claim 1, wherein the crude oil comprises less than 1% by weight phospholipid before addition of the gum phase.

3. A process according to claim 1 or claim 2, wherein the crude oil comprises less than 0.5% by weight phospholipid before addition of the gum phase.

4. A process according to any preceding claim, wherein the crude oil comprises at least 1% by weight phospholipid after addition of the gum phase.

5. A process according to any preceding claim, wherein the crude oil comprises at least 1.5% by weight phospholipid after addition of the gum phase.

6. A process according to any preceding claim, wherein the crude oil is obtained by pressing oil-containing plant seeds.

7. A process according to any preceding claim, wherein the crude oil comprises crude rapeseed (canola) oil.

8. A process according to any preceding claim, wherein the enzyme is contacted with the oil in the presence of 1 to 5% by weight water.

9. A process according to any of claims 3 to 8, wherein the degumming step comprises water degumming.

10. A process according any preceding claim, wherein the gum phase is separated from the oil by centrifugation.

11. A process according to any preceding claim, wherein the process does not comprise a step of clay treatment.

12. A process according to any preceding claim, wherein the enzyme comprises a chlorophyllase, pheophytinase, pyropheophytinase or pheophytin pheophorbide hydrolase.

13. A process according to any preceding claim, wherein the enzyme comprises a polypeptide sequence as defined in any one of SEQ ID NOs: 1 to 31, or a functional fragment or variant thereof.

14. A process according to claim 14, wherein the enzyme comprises a polypeptide sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1 to 31 over at least 50 amino acid residues.

15. A refined plant oil obtainable by a process as defined in any preceding claim.