WO2010143149A2 - Method - Google Patents

Abstract

The present invention provides methods and uses for treating pyropheophytin-containing compositions, particularly in order to remove pyropheophytin therefrom. The compositions are typically plant, algal, or bacterial derived products such as vegetable oils. The method comprises a step of contacting the compositions with an enzyme having pyropheophytinase activity. Also provided are related apparatus and products for performing such methods and uses.

Description

METHOD

FIELD

The present invention relates to a method, use, apparatus and related products for treating a composition comprising pyropheophytin. In one aspect, the invention is particularly applicable to the industrial processing of plant-derived food and feed products, such as vegetable oils. The invention may be employed to reduce or eliminate contamination by pyropheophytin in such products

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, degumrning, 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 entramment in the bleaching clay. Also the use of clay is expensive, this is particularly due to the treatment of the used clay (Le, the waste) which can be difficult, dangerous 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 26). 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 green colour and may be removed from the oil by an analogous process to that used for chlorophyll, for instance as described in WO 2006009676 by an esterase reaction catalyzed by an enzyme having a pheophytinase activity. Under certain conditions, some chlorophyllases are capable of hydrolyzing pheophytin as well as chlorophyll, and so are suitable for removing both of these contaminants. The products of pheophytin hydrolysis are the red/brown-colored pheophorbide and phytoL It is worth noting that pheophorbide can also be produced by the loss of a magnesium ion from chlorophyllide, i.e. following hydrolysis of chlorophyll (see Figure 26). WO 2006009676 teaches removal of pheophorbide by an analogous method to chlorophyllide, e.g, by aqueous extraction or silica adsorption. Notably, however, pheophorbide is less water soluble than cholrophyllide and therefore cannot be as easily washed out with aqueous extraction (in particular with water),

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, VoI, 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 lcnown as pyropheophorbide (see Figure 26). Pyropheophorbide is less polar than pheophorbide resulting in the pyropheophoribe having a decreased water solubility and an increased oil solubility compared with pheophorbide.

Pyropheophytin is 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. 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. Thus chlorophyll is a relatively minor contaminant compared to pyropheophytin (and pheophytin) in many plant oils,

Pyropheophytin has a green colour and is a major undesirable contaminant in the oil, in view of its adverse effects on both colour and stability. Despite the attention directed to chlorophyll and (to a lesser extent) pheophytin removal, there is still a need for a suitable method to remove pyropheophytin and its derivatives (e.g. pyropheophorbide) from compositions such as vegetable oils, In particular, the chlorophyllases described in the prior art typically have little or no pyropheophytinase activity and are thus incapable of removing pyropheophytin contamination.

SUMMARY

In one aspect the present invention provides a method for treating a pyropheophytin- containing composition, comprising contacting the composition with an enzyme which is capable of hydrolysing pyropheophytin.

Preferably the composition is derived from a plant, algae or bacteria. In one embodiment, the composition comprises a plant-derived oil, e.g. a vegetable oil. Preferably the composition comprises an oil selected from rice bran, soy, canola (rape seed), palm, olive, cottonseed, corn, palm kernel, coconut, peanut, sesame or sunflower oil

In one embodiment the enzyme comprises a pheophytinase or pheophytin pheophorbide hydrolase. The enzyme may be derived from, for example, a species selected from the following genera: Arabidopsis, Populus, Vitis, Ory∑a, Zea, Nicotiana, Ostreococcus,

Ostreococcus, Physcomitrella, Phaeodactylum, Chlamydomonas, or Micromonas or the enzyme may be derived from, for example, a species selected from Arabidopsis thaliana,

Populus trichocarpa, Vitis vinifera, Ory∑a sativa, Zea mays, Nicotiana tabacum, Osireococcus lucimarinus, Ostreococcm taurii, Physcomitrella patens, Phaeodactylum tricornuium, Chlamydomonas reinhardtii, or Micromonas sp RCC299.

Preferably the enzyme comprises an amino acid sequence selected from: LPGFGVG (SEQ ID NO: 13), DFLGQG (SEQ ID NO: 14), GNSLGG (SEQ ID NO: 15), LVKGVTLLNATPFW (SEQ ID NO: 16), HPAA (SEQ ID NO:17)₅ EDPW (SEQ ID NO: 18), and SPAGHCPH (SEQ ID NO: 19).

In one embodiment the enzyme comprises a polypeptide sequence as defined in SEQ ID NO:1 or any one of SEQ ID NO:s 4 to 12, or any one of SEQ ID NOs:21₅ 23 or 25, or a functional fragment or variant thereof, for example the enzyme comprises a polypeptide sequence having at least 50%, at least 75%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO:1 or any one of SEQ ID NO:s 4 to 12, or any one of SEQ ID NOs:21 ,

23 or 25, over at least 20, at least 50, at least 100 or at least 500 amino acid residues, or over the full length of the sequence.

In one embodiment, the enzyme has a pheophytinase to pyropheophytinase activity ratio of less than 80.

The enzyme may hydrolyze pyropheophytin in the composition to form pyropheophorbide. In some embodiments, the method further comprises a step of removing pyropheophorbide from the composition. Pyropheophorbide may be removed, for example, by a deodorization step or a silica treatment step, preferably by both a deodorization step and a silica treatment step.

Preferably the method comprises a two or more silica treatment steps. In one embodiment the silica treatment is performed at elevated temperature, e.g. at about 50 to 15O⁰C, at 70 to 110⁰C or at about 90⁰C.

In one embodiment the enzyme which is capable of hydrolysing pyropheophytin is immobilized on a solid support. The method may further comprising contacting the composition with an enzyme having chlorophyll ase activity, and the chlorophyllase may also optionally be immobilized on a solid support.

Preferably the method further comprises a step of contacting the composition with an acyl transferase. Preferably the concentration of pyropheophytin in the composition is reduced by at least 10%, at least 50%, at least 75% or at least 90% compared to the concentration of pyropheophytin present in the composition before treatment.

In another aspect, the present invention provides a process for refining a plant (e.g. vegetable) oil, comprising treating a pyropheophytin-containing plant oil with a pyropheophytinase. Such a process may be performed on an industrial scale and may comprise various method steps as desribed above. The process may further comprise steps typically used in vegetable oil processing, such as a hexane extraction and/or a degurnming step.

In a further aspect, the present invention provides use of a polypeptide having pyropheophytinase activity for removing pyropheophytin contamination from a composition. The use may be performed employing various method steps as discussed above.

In a further aspect, the present invention provides an apparatus for the enzymatic treatment of a pyropheopytin-containing composition, comprising (a) a plant oil refining apparatus; and (b) a polypeptide having pyropheophytinase activity operably integrated into the plant oil refining apparatus, such that the polypeptide is capable of hydrolyzing pyropheophytin in the composition during refining of the composition. The apparatus may comprise corresponding apparatus feature to perform a method or process as described above.

In a further aspect, the present invention provides a composition comprising a polypeptide having pyropheophytinase activity immobilized on silica. The polypeptide may be an enzyme as described in preferred embodiments of the method mentioned above,

In a further aspect, the present invention provides a polypeptide having an amino acid sequence as defined in SEQ 3DD NO;4, or encoded by a nucleic acid sequence as defined in SEQ ED NO:3, or a functional variant or fragment thereof having pyropheophytinase activity. For example, variants and/or fragments may show at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO:4 over at least 100, at least 200 or at least 300 amino acid residues or over the entire length of the sequence.

In a further aspect, the present invention provides a nucleic acid sequence as defined in SEQ ID NO:3, or a variant or fragment thereof encoding a functional pyropheophytinase. For example, variants and/or fragments may show at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO:3 over at least 300, at least 500 or at least 1000 nucleotide residues or over the entire length of the sequence. In further aspects the present invention provides an expression vector comprising a nucleic acid sequence as defined in SEQ ID NO:3

(e.g. an expression vector as shown in Figure 13) and a transformed (host) cell comprising such an expression vector-

In a further aspect, the present invention provides a polypeptide having an amino acid sequence as defined in SEQ ID NO:25, or a functional variant or fragment thereof having pyropheophytinase activity. For example, variants and/or fragments may show at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO:25 over at least 100, at least 200 or at least 300 amino acid residues or over the entire length of the sequence.

In a further aspect, the present invention provides a composition obtainable by the process or method as defined above. For example, the composition may be a plant, algal or bacterial product, particularly a refined plant oil, e.g. a refined vegetable oil

It has surprisingly been found that certain plant enzymes have pyropheophytinase activity, e.g. are capable of hydrolyzing an ester bond in pyropheophytin to form pyropheophorbide and phytol. Moreover, such pyropheophytinases are particularly useful for removing pyropheophytin contamination in plant-derived products such as vegetable oils.

BRIEF DESCRIPTION OF DRAWINGS

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

Figure 2 shows the results of HPLC analysis of sample 1 as defined in Table 5 (Control) before deodorization.

Figure 3 shows the results of HPLC analysis of sample 1 as defined in Table 5 (Control) after deodorization. Figure 4 shows the results of HPLC analysis of sample 2 as defined in Table 5 (comprising pheophorbide) before deodorization.

Figure 5 shows the results of HPLC analysis of sample 2 as defined in Table 5 (comprising pheophorbide) after deodorization-

Figure 6 shows the results of HPLC analysis of sample 3 as defined in Table 5 (comprising pyropheophorbide) before deodorization.

Figure 7 shows the results of HPLC analysis of sample 3 as defined in Table 5 (comprising pyropheophorbide) after deodorization.

Figure 8 shows the results of HPLC analysis of sample 4 as defined in Table 5 (comprising pheophytin) before deodorization.

Figure 9 shows the results of HPLC analysis of sample 4 as defined in Table 5 (comprising pheophytin) after deodorization.

Figure 10 shows the amino acid sequence of a pheophytin pheophorbide hydrolase (PPH) from Arabidopsis thaliana (SEQ ID NO:1). A chloroplast transit peptide is shown in bold.

Figure 1 1 shows the nucleotide sequence of a cDNA from Arabidopsis thaliana encoding pheophytin pheophorbide hydrolase (SEQ ID NO:2). The PPH of SEQ ID NO:1 is encoded by residues 173 to 1627 of SEQ ID NO:2.

Figure 12 shows a synthetic gene encoding pheophytin pheophorbide hydrolase (pheophytinase) with codons designed for expression in the filamentous fungi Trichoderma reesei (nucleotide sequence SEQ ID NO:3, amino acid sequence SEQ ID NO:4).

Figure 13 shows an expression construct containing a synthetic pheophytinase (PPH) gene fused to the catalytic core of cbhl via a kexin octapeptide linker.

Figure 14 shows SDS-PAGE of Trichoderma reesei transformants expressing PPH as a secreted protein in the culture supernatant.

Figure 15 shows the polypeptide sequence of Populus trichocarpa PPH (SEQ ID NO:5).

Figure 16 shows the polypeptide sequence of Vitis vinifera PPH (SEQ ID NO:6). Figure 17 shows the polypeptide sequence of Ricinus communis PPH (SEQ ID NO:7).

Figure 18 shows the polypeptide sequence of Oryza sativa (japonica cultivar- group) PPH (SEQ ID NO:8).

Figure 19 shows the polypeptide sequence of Zea mays PPH (SEQ ID NO:9).

Figure 20 shows the polypeptide sequence of Nicotiana tabacum PPH (SEQ ID NO:10).

Figure 21 shows the polypeptide sequence of Oiy∑a sativa Japonica Group PPH (SEQ ID NO: 11).

Figure 22 shows (a) the polypeptide sequence of Physcomitrella patens subsp. patens PPH (SEQ ID NO: 12) and (b) the polypeptide sequence of Λrabidopsis thaliana chlorophyllase (SEQ ID NO:20)

Figure 23 shows an amino acid sequence alignment of the Arabidopsis thaliana PPH protein with putative PPHs. Conserved blocks of the following amino acid sequences were found and are shown herein: LPGFGVG (SEQ ID NO:13), DFLGQG (SEQ ID NO: 14), GNSLGG (SEQ ID NO:Ϊ5), LVKGVTLLNATPFW (SEQ ID NO: 16), HPAA (SEQ ID NO: 17), EDPW (SEQ ID NO: 18), and SPAGHCPH (SEQ ID NO: 19).

Figure 24 is a diagrammatic representation of an oil refining process according to the present invention.

Figure 25 is a diagrammatic representation of a vegetable oil refining process and apparatus according to the present invention.

Figure 26 shows the reactions involving chlorophyll and derivatives and enzymes used in the present invention.

Figure 27 shows the amino acid sequence of Triticum aestivum chlorophyllase (SEQ ID NO:21).

Figure 28 shows a nucleotide sequence encoding Triticum aestivum chlorophyllase (SEQ ID NO:22),

Figure 29 shows the amino acid sequence of Chlamydomonas reinhardtii chlorophyllase (SEQ ID NO:23). Figure 30 shows a nucleotide sequence encoding Chlamydomonas reinhardtii chlorophyllase

(SEQ ID NO:24).

Figure .31 shows schematically the fusion of the wheat (Triticum aestivum) chlorophyllase gene to the aprE signal sequence,,

Figure 32 shows schematically the plasmid pBN-TRI_CHL containing the wheat (Triticum aestivum) chlorophyllase gene.

Figure 33 shows schematically the fusion of the Chlamydomonas reinhardtii chlorophyllase gene to the aprE signal sequence.

Figure 34 shows schematically the plasmid pBN-CHL_CHL containing the Chlamydomonas reinhardtii chlorophyllase gene.

Figure 35 shows an amino acid sequence of a variant (Triticum NdI -16) of Triticum aestivum chlorophyllase lacking the N-terminal 16 amino acids compared to the wild-type enzyme (SEQ ID NO:25).

Figure 36 shows a nucleotide sequence encoding a variant (Triticum NdI -16) of Triticum aestivum chlorophyllase lacking the N-terminal 16 amino acids compared to the wild-type enzyme (SEQ ID NO:26).

DETAILED DESCRIPTION

In one aspect the present invention relates to a method for treating a pyropheophytinase- containing composition. Typically the method is performed in order to remove pyropheopytin from the composition, or to reduce the level of pyropheopytin in the composition, for instance where the pyropheophytin is present as a contaminant.

Pyropheopytin has a green colour, which is derived from the porphyrin (chlorin) ring present in the molecule. Thus the presence of pyropheophytin in a composition, e.g. a vegetable oil, can give such a composition an undesirable green or greenish colour. In one embodiment, the present method may be performed in order to remove or reduce the green colouring present in a composition. Accordingly the present method may be referred to as a bleaching or decolorizing process. Hydrolysis of pyropheophytin yields phytol and pyropheophorbide (see Figure 26),

Pyropheophorbide contains the colour-bearing porphyrin ring, although loss of the phytol chain means that pyropheophorbide has a reddish brown, rather than green, colour. In some embodiments, it may also be desirable to remove pyropheophorbide and to reduce the red/brown colouring in the composition. Thus in one embodiment of the invention, the method may further comprise a step of removing or reducing the level of pyropheophorbide in the composition. The present method may involve bleaching or de-colorizing to remove the green and/or red/brown colouring of the composition.

The present invention in one embodiment relates to the selection, expression and use of an enzyme with activity on magnesium free degradation products of chlorophyll (in particular at least pyropheophytin, such as pheophytin and pyropheophytin) and with low or no activity on chlorophyll. Preferable the enzyme of the invention has 80% more activity on magnesium free chlorophyll degradation products (preferably on pyropheophytin) than on chlorophyll, more preferable the enzyme of the invention has more than 90% more activity on magnesium free chlorophyll degradation products (preferably on pyropheophytin) than on chlorophyll. More preferable 95% more activity on magnesium free chlorophyll degradation products

(preferably on pyropheophytin) than on chlorophyll..

Compositions

Any composition comprising pyropheophytin may be treated according to the present method, in order to remove undesirable pyropheophytin contamination. Preferably the composition is a plant-derived preparation, an algal preparation or a bacterial-derived preparation, e.g. the composition is a product derived from any type of plant, algae or bacteria (e.g. cyanobacteria), In one embodiment the composition comprises a plant material, plant oil or plant extract The term "plant" includes whole plants, plant parts (e.g., leaves, stems, flowers, roots, etc), 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 gymnosperrns. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous states.

In preferred embodiments, the composition may comprise a plant oil such as 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 methods 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.

Alternatively, the composition may comprise an algal preparation; a textile, thread or fabric or a cloth; or a wood or paper product by-product, such as a wood pulp, a paper pulp, a Kraft pulp, or a non-wood paper product or by-product, such as a rice paper. In other aspects of the methods, the composition may comprise a pharmaceutical or cosmetic formulation (e.g. liposomes for pharmaceuticals and cosmetics), a biodiesel oil, a food, an edible oil, a feed, or a dietary supplement

The methods of the invention can be used to treat crude or refined oils derived from plant (e.g. vegetable or algae) sources or alternatively from synthetic sourceSn The method of the invention can be used to treat crude or refined oils at higher oil concentrations, or, in one aspect, used to treat unrefined and non-diluted crude oils. The methods of the invention can be used in conjunction with methods for processing high phosphorus oils (e.g. a soy bean oil).

Pyropheophytin removal

The pyropheophytin may be present in the composition (e.g. a preparation, feed, food or oil) naturally, as a contaminant, or as an undesired component in a processed product. The pyropheophytin may be present at any level in the composition. Typically pyropheophytin may be present as a natural contaminant in the composition (e.g. in a vegetable oil) at a concentration of 0.001 to 1000 mg/kg (0.001 to 1000 ppm, 10^"7 to 10^"s wt %), based on the total weight of the composition (e.g. a vegetable oil). In further embodiments, the pyropheophytin may be present in the composition at a concentration of 0.1 to 100, 0,5 to 50, 1 to 50, 1 to 30 or 1 to 10 mg/kg, based on the total weight of the composition.

The method of the present invention typically reduces the level of pyropheophytin in the composition. For example, the method may reduce the concentration of 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 pyropheophytin (by weight) present in the total composition before treatment. Thus in particular embodiments, the pyropheophytin concentration in the composition (e.g. in a vegetable oil) after treatment may be less than 100, less than 50, less than 30, less than 10, less than 5, less than I₅ 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).

Pyropheophorbide removal

The method of the present invention may optionally involve a step of removing pyropheophorbide. Pyropheophorbide may be present in the composition due to the hydrolysis of pyropheophytin 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 fiom chlorophyllide following the action of chlorophyllase on chlorophyll (see Figure 26). Various steps in the formation of pyropheophorbide may be favoured by the processing conditions used in oil refining, in particular heat, or by the use enzymes such as pheorphorbidase, chlorophyllase and/or pheophytinase in the process

The pyropheophorbide may be present at any level in the composition. Typically pyropheophorbide may be present in the composition (e.g. in a vegetable oil), either before or after treatment with a pyropheophytinase 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 composition (e.g., a vegetable oil). In further embodiments, the 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.

In one embodiment the method of the present invention reduces the level of pyropheophorbide in the composition, compared to either or both of the levels before and after pyropheophytinase treatment. Thus in some embodiments the pyropheophorbide concentration may increase after pyropheophytinase treatment. Typically the method involves a step of removing pyropheophorbide such that the pyropheophorbide concentration is lower than after pyropheophytinase treatment. Preferably the pyropheophorbide produced by this enzymatic step is removed from the composition, such that the final level of pyropheophorbide in the composition is lower than before pyropheophytinase treatment.

For example, the method may reduce the concentration of 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 pyropheophorbide (by weight) present in the total composition before the pyropheophorbide removal step, e.g. before or after pyropheophytinase treatment. Thus in particular embodiments, the pyiopheophorbide concentration in the composition (e.g. in a vegetable oil) after the pyropheophorbide 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)-

Pyropheophytinase

The method of the present invention comprises a step of contacting a pyropheophytin- containing composition with an enzyme having pyropheophytinase activity. Any polypeptide having an activity that can modify pyropheophytin can be used as the enzyme in the method of the invention. By "pyropheophytinase activity" it is preferably meant that the enzyme can hydrolyse an ester bond in pyropheophytin to produce phytol and pyropheophorbide. Thus the enzyme typically has an esterase or hydrolase activity. Preferably the enzyme is capable of pyropheophytinase activity in an oil phase, and optionally also in an aqueous phase.

Pyropheophytinase activity may be detected using any suitable assay technique, for example based on the enzyme activity (pheophytinase and phyropheophytinase activity) assay described in the examples below using pyropheophytin as substrate. For example, pyropheophytinase activity may be detected using fluorescence-based techniques, e.g. by monitoring pyropheophorbide as described in Example 5 below. In one suitable assay, a polypeptide to be tested for pyropheophytinase activity is incubated in the presence of pyropheophytin, and pyropheophytin, pyropheophorbide and/or phytol levels are monitored by fluorescence measurement. Alternatively, a pyropheophytinase assay may be based on HPLC detection and quantitation of pyropheophytin, pyropheophorbide and/or phytol levels following addition of a putative enzyme, e.g. based on the techniques described in the examples with particular reference to Example 5 below,

Pyropheophytinase activity may be determined using methods based on those disclosed in, for example, AIi 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. Pyropheophytin may be used in place of chlorophyll as substrate where appropriate. Alternatively, a suitable assay may be based on HPLC detection and quantitation of pyropheophytin or pyropheophorbide levels following addition of a putative enzyme, e.g. based on the techniques described below. In one embodiment, the assay may be based on a method as described in Homero-Mendez et al. (2005), Food Research International 38(8-9): 1067-1072, In another embodiment, the following assay may be used:

170 μl mM HEPES, pH 7.0 is added 20 μl OJ mM pyropheophytin dissolved in acetone. The enzyme is dissolved in 50 mM HEPES, pH 7.0. 10 μl enzyme solution is added to 190 μl substrate solution to initiate the reaction and incubated at 40 ⁰C for various time periods. The reaction was stopped by addition of 350 μl acetone. Following centrifugation (2 min at 18,000 g) the supernatant was analyzed by HPLC, and the amounts of pyropheophytin and pyropheophorbide determined.

One unit of pyropheophytinase activity is defined as the amount of enzyme which hydrolyzes one micromole of pyropheophytin per minute at 40⁰C, e.g. in an assay method as described herein.

In preferred embodiments, the enzyme used in the present method has pyropheophytinase activity of at least 100 U/g, at least 250 U/g or at least 500 U/g, based on the units of activity per gram of the purified enzyme, e.g. as determined by an assay method described herein.

In some embodiments, the enzyme may have further activities in addition to the pyropheophytinase activity, e.g. a pheophytinase activity and/or a chlorophyllase activity. Thus the enzyme need not be selective for pyropheophytin, and may be capable of utilizing pheophytin and/or chlorophyll as substrates in addition to pyropheophytin, provided that the enzyme shows significant activity towards pyropheophytin, By "enzyme" it is intended to encompass any polypeptide having pyropheophytinase activity, including e.g. catalytic antibodies, enzyme fragments, etc. Any isolated, recombinant or synthetic or chimeric (or a combination of synthetic and recombinant) polypeptide (e.g. enzyme or catalytic antibody) can be used. Thus as used herein, the term "pyropheophytinase" encompasses any polypeptide capable of hydrolyzing pyropheophytin. Pheophytinase and chlorophyllase activity may be determined by analogous methods to those described above for pyropheophytinase, replacing pyropheophytin as substrate with pheophytin or chlorophyll where appropriate. In one embodiment, the enzyme is capable of hydrolyzing pheophytin and pyropheophytin

More preferably, the enzyme is capable of hydrolyzing pheophytin and pyropheophytin but is incapable of hydrolyzing chlorophyll. For example, the enzyme may be pheophytinase or pheophytin pheophoibide hydrolase (PPH), e.g. an enzyme as described in Schelbert et al, The Plant Cell 21 :767-785 (2009).

In another embodiment, the enzyme preferably has a pheophytinase to pyropheophytinase activity ratio of less than 80, less than 70, less than 6O₅ less than 50, less than 40 or less than 30, For example, the enzyme may have a pheophytinase to pyropheophytinase activity ratio of 0,1 to 70, 1 to 50 or 10 to 30. The pheophytinase to pyropheophytinase activity ratio may be calculated by determining pheophytinase activity and pyropheophytinase activity using methods described above, and dividing the pheophytinase activity by the pyropheophytinase activity. Particularly preferred enzymes having a low ratio of pheophytinase to pyropheophytinase activity are derived from Triticum sp or Chlamydomonas sp. as described below.

In one embodiment, the enzyme is derived from wheat, e.g. from Triticum sp , especially from Triticum aestivum. For example, the enzyme may be a polypeptide comprising the sequence of SEQ ID NO:21 (see Figure 27), or may be encoded by the nucleotide sequence of SEQ ID NO:22 (see Figure 28).

In another embodiment, the enzyme is derived from Chlamydomonas sp , especially from Chlamydomonas reinhardtii. For example, the enzyme may be a polypeptide comprising the sequence of SEQ ID NO:23 (see Figure 29), or may be encoded by the nucleotide sequence of SEQ ID NO:24 (see Figure 30).

In one embodiment, the enzyme is an N-terminally truncated variant of a chlorophyϊlase and/or pyropheophytinase, e.g. an N-terminally truncated variant of SEQ ID NO:1, any one of SEQ ID NO:s 4 to 12, or SEQ ID NOs:21 or 23. In particular embodiments, such N- terminally truncated variants may lack at least 1, 2, 5, 10 or 15 amino acids (e.g. 1 to 30 or 5 to 20 amino acids) at the N-terminal compared to the parent sequence. In one embodiment, the enzyme comprises the sequence of SEQ ID NO:25 (see Figure 35), i.e. an N-terminally truncated variant of SEQ ID NO:21.

It has surprisingly been found that chlorophyllases from Triticum and Chlamydomonas have pyropheophytinase activity, and a relatively low ratio of pheophytinase to pyropheophytmase activity- Moreover, an N-terminally truncated variant of Triticum chlorophyllase has a reduced ratio of pheophytinase to pyropheophytϊnase activity compared to the wild-type enzyme.

Pheophytin pheophorbide hydrolase

It has surprisingly been found that PPH and related enzymes are capable of hydrolyzing pyropheophytin in addition to pheophytin. However PPH is inactive on chlorophyll. As described in Schelbert et ctl , PPH orthologs are commonly present in eukaryotic photosynthesizing organisms. PPHs represent a defined sub-group of α/β hydrolases which are phylogenetically distinct from chlorophyllases, the two groups being distinguished in terms of sequence homology and substrates.

In specific embodiments of the invention, the enzyme may be any known PPH derived from any species or a functional variant or fragment thereof or may be derived from any known PPH enzyme. For example, in one embodiment, the enzyme is a PPH from Arabidopsis thaliana, e.g. a polypeptide comprising the amino acid sequence of SEQ ID NO:1, or a polypeptide encoded by the nucleotide sequence of SEQ ID NO:2 (NCBI accession no. NPJ 96884, GenBank ID No. 15240707), or a functional variant or fragment thereof In another embodiment, the enzyme comprises the amino acid sequence of SEQ ID NO:4, or a polypeptide encoded by the nucleotide sequence of SEQ ID NO:3, or a functional variant or fragment thereof.

In further embodiments, the enzyme may be a PPH derived from any one of the following species: Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera, Oiyza sativa, Zea mays, Nicotiana tabacum, Ostreococcus lucimarinus, Ostreococcus taurii, Physcomitrella patens, Phaeodactylum ti'icornutum, Chlamydomonas reinhardtii, or Micromonas sp. RCC299, For example, the enzyme may be a polypeptide comprising an amino acid sequence, or encoded by a nucleotide sequence, defined in one of the following database entries shown in Table 1 , or a functional fragment or variant thereof:

Table 1

Organism Accession Genbank ID Arabidopsis thaliana NPJ 96884 15240707 Populus trichocarpa XPJ)O 2314066 224106163 Vitis vinifera CAO40741 157350650 Oryza sativa (japonica) NPJ)01057593 115467988 Zea mays ACF87407 194706646 Nicotiana tabacum CAO99125 156763846 Ostreococcυs lucimarinus XPJXH415589 145340970 Ostreococcus tauri CAL50341 116000661 Physcomitrella patens XPJ)01761725 168018382 Pliaeodactylum tricomutum XP_002181821 219122997 Chlarnydomonas reinhardtii XPJ)01702982 159490010 Micromonas sp. RCC299 ACO62405 226516410

For example, the enzyme may be a polypeptide as defined in any of SEQ ID NO:s 5 to 12, or a functional fragment or variant thereof

Variants and fragments

Functional variants and fragments of known pyropheophytinase (e.g. PPH) sequences may also be employed in the present invention. By "functional" it is meant that the fragment or variant retains a detectable pyropheophytinase activity. Typically such variants and fragments show homology to a known pyropheophytinase (e.g. PPH) 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 pyropheophytinase (e.g. PPH) amino acid sequence, e.g. to SEQ ID NO:1 or any one of SEQ ID NOs: 4 to 12 or a sequence defined in Table 1, or to SEQ ID NO:21 , 23 or 25, over a region of at least about 10, 2O₅ 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 pyropheophytinase activity suitable for use in the methods of the invention may be identified by determining the presence of conserved sequence motifs present e.g. in known PPH sequences. Conserved sequence motifs include the following: LPGFGVG (SEQ ID NO:13), DFLGQG (SEQ ID NO:14), GNSLGG (SEQ ID NO:15), LVKGVTLLNATPFW (SEQ ID NO: 16), HPAA (SEQ ID NO: 17), EDPW (SEQ ID NO: 18), and SPAGHCPH (SEQ ID NO: 19). Thus preferred pyropheophytinases for use in the present invention comprise one or more of these sequences. The GNSLGG (SEQ ID NO: 15) motif contains the active site serine residue. It is particularly preferred that the enzyme used in the method of the invention comprises a GNSLGG sequence. Polypeptide sequences having suitable pyropheophytinase 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 pyropheophytinases

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 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 SX. 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 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 pyropheophytinases 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 1, 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 pyropheophytinases with preferred characteristics. Suitable methods for performing 'shuffling' can be found in EP0752008, EPl 138763, EPl 103606. Shuffling can also be combined with other forms of DNA mutagenesis as described in US 6,180,406 and WO 01/34835.

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

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 a pyropheophytinase (e.g. a PPH) used in the invention may encode a variant pyropheophytinase (e.g. a variant PPH), i.e. the pyropheophytinase (e.g. PPH) 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 pyropheophytinase activity.

Pyropheophytinase polypeptide sequences

The present invention also encompasses the use of amino acid sequences encoded by a nucleotide sequence which encodes a 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 μl 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

50⁰C following overlay with nitrogen and addition of 5 μl of 45 mM dithiothreitol. After cooling to room temperature, 5 μl 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 μl of water and 5 μg of endoproteinase Lys-C in 5 μl of water may be added to the above reaction mixture and the digestion may be carried out at 37⁰C under nitrogen for 24 hours. The resulting peptides may be separated by reverse phase HPLC on a VYDAC CIS column (0,46x15cm; lOμm; 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-chromato graphed on a Develosil Cl 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 (Le, 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, talcing into consideration gap penalties, A suitable computer program for carrying out such an alignment is the Vector NTI Advance™ 1 1 (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:

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 L

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:

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 Le. 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 α-carbon substituent group is on the residue's nitrogen atom rather than the α-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RI 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 phosphorolhioate 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 cairied 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 PiIeUp 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. Pyropheophytinase formulation and dosage

Enzymes used in the methods of the invention, e.g. a pyropheophytinase, 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 composition with a pyropheophytinase, 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, In one embodiment, an enzyme having pyropheophytinase activity is added to the composition, e.g. a vegetable oil.

The pyropheophytinase may be dosed into the composition, e.g. a vegetable oil, in any suitable amount. For example, the enzyme may be dosed in a range of about 0,0001 to lU/g of the composition, preferably 0,001 to 0.1 U/g, e.g, 0.005 to 0.01 U/g of the composition (e.g. vegetable oil). Notably one unit is defined in accordance with the "enzyme activity (pheophytinase and phyropheophytinase activity) assay" taught in the Examples below.

The method of the invention can be practiced with immobilized enzymes, e.g. an immobilized 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 pheophytinase 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. Enzyme reaction conditions

Suitably the composition (e.g. vegetable oil) may be incubated (or admixed) with the pyropheophytinase between about 5°C to and about 100⁰C, more preferably between 10⁰C to about 90⁰C, more preferably between about 15⁰C to about 8O⁰C, more preferably between about 20°C to about 75°C, more preferably at about 30 to about 60°C, preferably at about 45 to about 55°C In another embodiment, suitably the method and/or use according to the present invention may be carried out at below about 6O⁰C, preferably below about 50°C, preferably below about 4O⁰C, Preferably the temperature of the composition (e.g. vegetable oil) may be at the desired reaction temperature when the enzyme is admixed therewith.

The composition (e.g. vegetable 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 composition (e.g. vegetable oil).

Suitably the reaction time (Le, the time period in which the admixture is held), preferably with agitation, is for a sufficient period of time to allow hydrolysis of pyropheophytin to form pyropheophorbide and phytol. 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 pH6.5, more preferably between about pH 6.5 and about pH 7.5, 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.

Suitably the water content of the composition (e.g. vegetable oil) when incubated (or admixed) with the pyropheophytinase 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%. When an immobilised enzyme is used, suitably the water activity of the immobilised enzyme may be in the range of about 02 to about 0.98, preferably between about 0-4 to about 0.9, more preferably between about 0.6 to about 0,8.

Processing and refining of plant (vegetable) oils

In one embodiment, the method of the present invention may be used in the enzymatic processing of plant-derived oilSn In a typical vegetable oil processing method, oil is extracted in hexane, the crude vegetable oil is degummed, (optionally, caustic neutralized), bleached using, e.g. clay adsorption with subsequent clay disposal, and deodorized to produce refined, bleached and deodorized or RBD oil. The need for the degumming step depends on phosphorus content and other factors (all known in the art).

In embodiments of the present invention, an enzymatic reaction involving application of the pyropheophytinase may be performed at various stages of this method. For example, the pyropheophytinase may be contacted with the crude oil, during a degumming step, or in a bleaching step (e.g. in place of clay bleaching) following degumming.

Silica treatment

Thus in one embodiment of the invention, the process comprises degumming a crude vegetable oil, bleaching using an enzyme having pyropheophytinase activity, followed by deodorization. Preferably the method comprises a step of silica treatment, especially subsequent to the pyropheophytinase 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).

Unlike chlorophyllide, pyropheophorbide (and pheophorbide) are less water soluble and are not particularly suited to removal by an aqueous extraction step. The silica treatment step is particularly suitable for removing pyropheophorbide (and also pheophorbide) produced by the action of the pyropheophytinase, Pyropheophorbide (and pheophorbide) may also be removed by the deodorization step. Preferably the process comprises both a deodorization step and a silica treatment step.

More preferably 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 is preferably performed at elevated temperature, e.g. at above about

3O⁰C, more preferably about 50 to 150⁰C₅ about 70 to HO⁰C, about 80 to 100⁰C or about 85 to 95°C , most preferably about 90⁰C,

Degumming and Oil Refining

The methods can be used in conjunction with processes based on extraction with hexane and/or with subsequent refining of the crude extracts to edible oils. For instance the methods of the present invention can be used in conjunction with enzyme assisted oil extraction (see Journal of Americal Oil Chemists' Society (2006), 83 (11), 973-979). The first step in the refining sequence is the so-called "degumming" process, which serves to separate phosphatides by the addition of water. The material precipitated by degumming is separated and further processed to mixtures of lecithins . The commercial lecithins, such as soybean lecithin and sunflower lecithin, are semi-solid or very viscous materials. They consist of a mixture of polar lipids, mainly phospholipids, and oil, mainly triglycerides. The methods of the invention can be used before or after any step in this process, or before or after any combination of steps, or before or after all of the steps, in a process, e.g., prior to, during or following mechanical and/or chemical extraction, degumming and/or bleaching and the like.

The methods of the invention can be used in any degumming procedure, including water degumming, ALCON oil degumming (e.g., for soybeans), safmco degumming, "super degumning," UF degumming, TOP degumming, urn^" -de gumming, 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.

The methods of the invention can be used in any oil processing method, e.g., degumming or equivalent processes. For example, methods of the invention can be used in processes as described in U.S. Patent Nos. 5,558,781 ; 5,288,619; 5,264,367; 6,001,640; 6,376,689; WO 0229022; WO 2006009676; oil degumming as described, e.g., in 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. Further oil processing steps

Following an enzymatic treatment step using a pyropheophytinase according to the present invention, in one embodiment the treated liquid (e.g. oil) is separated with an appropriate means such as a centrifugal separator and the processed oil is obtained. Upon completion of the enzyme treatment, if necessary, the processed oil can be additionally washed with water or organic or inorganic acid such as, e.g., acetic acid, phosphoric acid, succinic acid, and the like, or with salt solutions.

Methods of the invention also can be practiced using processes as described in U.S. Patent No. 5,315,021. For example, in one aspect, the methods of the invention can be practiced with processes for removing color impurities other than pyropheophtyinase from vegetable oils, for instance for removing chlorophyll, pheophytin and derivatives thereof. For instance in one embodiment, the processes can comprise dispersing a source of phosphoric acid in vegetable oil to form a mixture having a moisture content of less than 0.1% by weight which mixture is maintained at a temperature in the range of 70⁰C to 16O⁰C until a precipitate containing chlorophyll color impurities is formed. This can be followed by separating the precipitated material from the oil to remove the chlorophyll color impurities with the precipitated material, e.g. during conventional oil processing up to and including the removal of bleaching clay from the oiL

Chlorophyllase treatment

In one embodiment, the method (e.g. a vegetable oil processing method) may fiirther comprise a step of contacting the composition with a chlorophyllase, for example using a method as described in WO 2006009676. For example, in one aspect, the method may comprise a step of enzymatically treating chlorophyll- containing or chlorophyll-contaminated compositions by hydrolyzing chlorophyll to phytol and chlorophyllide. Any chlorophyllase, chlase or chlorophyll chlorophyllido-hydrolyase or polypeptide having a similar activity (e.g., chlorophyll-chlorophyllido hydrolase 1 or chlase 1, or, chlorophyll-chlorophyllido hydrolase 2 or chlase 2, see, e.g. NCBI P59677-1 and P59678, respectively) can be used in this step of the method of the invention.

Any polypeptide that catalyses the hydrolysis of a chlorophyll ester bond to yield chlorophyllide and phytol can be used in this step of the method of the invention. 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. For example, the Arabidopsis thaliana chlorophyllase can be used as described, e,g. in NCBI entry NM_123753. Thus the chlorophyllase may be a polypeptide comprising the sequence of SEQ ID NO:20 shown in Figure 22b. In another embodiment, the chlorophyllase is derived from algae, e.g. from Phaeodactylum (ricornutum.

As described in WO 2006009676, chlorophyllide may be removed by an aqueous separation step and/or silica treatment. The method can further comprise modifying pH (e.g. increasing pH) to promote aqueous separation of chlorophyllide. Thus, the methods of the invention can also comprise a caustic neutralization processes, e g., with caustic-neutralized pH conditions. In one aspect, the compositions and methods of the invention comprise a neutralization step.

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. α-amylases), glucanases, polygalacturonases, galactolipases, cellulases, hemicellulases, pectinases and other plant cell wall degrading enzymes, as well as mixed enzyme preparations and cell Iy sates. 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.

In one aspect, the methods of the invention can be practiced with methods as described in U.S. Patent No. 6,376,689. For example, in one aspect, the compositions and methods of the invention can comprise a single-step acid degumming/decolorizing process that removes pyropheophytm,, and optionally pyropheophytm derivatives, pheophytϊn, chlorophyll and related compounds, from vegetable oils from seeds, especially frost damaged seeds which have large amounts of chlorophyll-type compounds. In one aspect, the methods of the invention further comprise a mixture of aqueous sulfuric and phosphoric acids that is blended with the oil to remove chlorophyll-type compounds from the oil. Plant oil processing apparatus and methods

In a further aspect, the present invention provides an apparatus for the enzymatic treatment of pyiopheophytin-containing compositions comprising (a) a plant (e.g. vegetable) oil refining apparatus; and (b) a polypeptide having a pyropheophytinase activity operably integrated into the plant (e.g. vegetable) oil refining apparatus. Typically the polypeptide having pyropheophytinase activity is integrated into the apparatus such that it is capable of hydrolyzing pyropheophytin during processing of the pyropheophytni-containing composition (e.g. a vegetable oil).

The apparatus may comprise any suitable vegetable oil refining apparatus or combination thereof, e.g. an oil leaving expellor (e.g. from Pennwalt Corp.), or a gravitational gum separation device. The apparatus may comprise immobilized enzymes, e.g., an immobilized pyropheophytinase and optionally an immobilized chlorophyllase. For example the apparatus may comprise a silica-immobilized pyropheophytinase. In one embodiment the silica comprises a silica gel or equivalent, e.g. a TriSyl Silica or a SORBSIL R™ silica. The apparatus may further comprise means for adjusting pH, e.g. increasing pH (caustic treatment), and then, alternatively, neutralizing pH.

In one embodiment, the method of the present invention may be performed in an oil refining process exemplified by Figure 24. The first stage of this process comprises hexane extraction to form crude oil, followed by water degurnrnmg, caustic neutralization, bleaching, silica treatment and deodorization. As shown in Fig. 24, pyropheophytinase treatment may be applied at various stages of this process, including to the crude oil, during water degurnming, or before silica treatment. The degumming can be a "traditional" or an enzymatic degurnming, e.g. involving phospholipid hydration and/or hydrolysis.

In the method as exemplified by Figure 24, a chlorophyllase may also be used, and the chlorophyllase treatment may take place at the same stage as pyropheophytinase treatment or at a different stage. The pyropheophytinase (and optional chlorophyllase) treatment may be considered to be an enzymatic bleaching step, and may replace a traditional clay adsorption bleaching step. In one aspect, the exemplary process of the invention comprises a subsequent aqueous separation step to remove the reaction products of chlorophyllase treatment, gum and/or soap. In an alternative embodiment, the pyropheophytinase treatment may be applied to an oilseed preparation before performing the processing steps shown in Figure 24, i.e. before hexane extraction.

Figure 25 illustrates another embodiment of a method and apparatus according to the present invention. Figure 25 illustrates various aspects of a vegetable oil refining apparatus, including components such as a degumming tank, static mixer, day tank, caustic tank, strainer, retention mixers, refining centrifuge, water wash centrifuge and vacuum dryer. The enzyme of the present invention having pyropheophytinase activity can be used in one or several or all of the following steps: added to the crude oil, in the degumming process or in the degummed oil, added to a storage or holding tank, a caustic tank, and/or a retention mixer. For example, in particular embodiments pyropheophytinase immobilized on silica may be added to the day tank or retention mixers.

The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples.

Examples

In the following examples, a pheophytinase from Arabidopsis thaliana is expressed by recombinant DNA methods and a pheophytinase enzyme is isolated which has activity on both pheophytin and pyropheophytin in vegetable oils. Pheophorbide and pyropheophorbide produced in the oil by pheophytinase/pyropheophytinase activity can then be removed by steps such as adsorption to silica and/or by deodorization. Silica is very efficient in removing the polar degradation products pheophorbide and pyropheophorbide.

Materials

The enzyme used is pheophytinase from Arabidopsis thaliana, (At PPH), GenBank ID 15240707, in an aqueous solution containing 5 Units/ml pheophytinase,

Pheophytinase is immobilized on silica accoiding to the following procedure. 3,0 g silica, (Sigma S5505 or Trisyl 300 from Grace Davison) is added to 6 ml of the obtained pheophytinase (cell extract) and stirred for 2 hours at room temperature. The silica is recovered by 10 minutes centrifugation at 300Og, The pelleted silica is washed twice with demineralized water. Crude rape seed oil from Scanola DK, containing 7 ppm pheophytm and 5 ppm pyropheophytin is used as the oil.

Pheophytin is produced from spinach chlorophyll by acid treatment according to "Extraction, Purification, and Characterization of Chlorophylls from Spinach Leaves." Journal of Agricultural and Food Chemistry 40.2 (1992): 215-220, Pheophytin (0.98 mg/ml) is dissolved in acetone.

Pyropheophytin (0,5 mg/ml) is produced from pheophytin dissolved in pyridine by heat treatment at 100⁰C according to "Determination of chlorophylls and their derivatives in Gynostemma pentaphyllurn Makino by liquid chromatography-mass spectrometry,", Journal of Pharmaceutical and Biomedical Analysis 48.1 (2008); 105-12. Pheophorbide and pyropheophorbide are obtained from Frontier Scientific.

Analytical methods

HPLC analysis is performed according to the following conditions, and generally as described in "Determination of chlorophylls and carotenoids by high-performance liquid chromatography during olive lactic fermentation", Journal of Chromatography, 585, 1991, 259-266,

Stock solutions of chlorophylls A and B are prepared as follows. 5 mg Chlorophyll A, from Anacystis nidulans algae, is dissolved in 5 ml acetone, 5 mg Chlorophyll B, from spinach (Sigma Life Science 25740) is dissolved in 5 ml acetone. Each chlorophyll solution is transferred to a 5 ml volumetric flask, distributed to 10 vials with 500μl in each, and stored frozen. 200 μl of each stock solution of chlorophylls A and B is added to 20 ml acetone, and 1 μl of the resultant solution added to 10 ml acetone to provide a control solution for HPLC.

Pheophytin in the oil samples was concentrated according to a solid-phase extraction procedure performed generally as described in "Routine and sensitive SPE-HPLC method for quantitative determination of pheophytin a and pyropheophytin a in olive oils", Food Research International, 38, 2005, 1067-1072, using Sep-Pak® *Plus tC18 Environmental Cartridges (WAT036800), 900mg, 17% Carbon Load.

Two tC18 cartridges are fitted in tandem (2x900mg₅ hold-up volumes -3 ml). The tClS cartridges are conditioned with 5 ml petroleum ether (40-60⁰C quality), without allowing the column to dry. A 1 ml oil sample is drawn into a 2 ml disposable syringe and pressed sample through the tC18 cartridges. The cartridges are washed with 12 ml petroleum ether (using a 10 ml disposable syringe).

The chlorophyll derivatives are eluted with 6 ml acetone (using a 5 ml disposable syringe). The acetone solution is evaporated to dryness under nitrogen. The temp is kept at 50-60⁰C. The extracted sample is reconstituted in 0.5 ml acetone and centrifuged at 4500rpm/3260rcf for 5 minutes. The sample is transferred to a vial with insert and analysed,

Pheophorbide and pyropheophorbide in the oil samples were concentrated according to a solid-phase extraction procedure performed using 1ST Isolute™ SPE Columns, (460-0050-B), Silica, SOOmg. The method is performed essentially as for tC18 cartridges except that cyclohexane is used in place of petroleum ether.

An example of a representative HPLC chromatogram (from Journal of Chromatography, 585, 1991, 259-266) and the assignment of peaks are given in Figure 1.

Enzyme activity (pheophytinase or pyropheophytinase activity) assay

170 μl mM HEPES, pH 7.0 is added 20 μl 0.3 mM pheophytin or pyropheophytin dissolved in acetone. The enzyme is dissolved in 50 mM HEPES, pH 7.0. 10 μl enzyme solution is added to 190 μl substrate solution to initiate the reaction and incubated at 25 ⁰C for various time periods. The reaction was stopped by addition of 350 μl acetone. Following centrifugation (2 rnin at 18,000 g) the supernatant was analyzed by HPLC, and the amounts of pheophytin and pheophorbide or pyropheophytin and pyropheophorbide determined. One unit of enzyme activity for pheophytinase activity is defined as one mϊcromole of hydrolyzed pheophytin per minute, One unit of enzyme activity for pyropheophytinase activity is defined as one micromole of hydrolyzed pyropheophytin per min.

Example 1

Expression of Pheophytin Pheophorbide Hydrolase (Pheophytinase) in Trichoderma reesei

The amino acid sequence (Protein accession BAH19780) encoded by the gene AT5G13800 from Arabidopsis thaliana, was retrieved from the NCBI sequence database: httρ://www.ncbi,nlrn.nih.gov/sites/entrez?db=protem&cmd=searchc&terrn=^:AT5G 13800. The sequence is shown in Figure 10 (SEQ ID NO:1). The AT5G13800 gene encodes a protein of 484 amino acids in length consisting of the mature protein of 438 amino acids and an N- terminal chloroplast transit peptide of 46 residues. The chloroplast transit peptide is shown in bold in Figure 10. The protein has been characterized as a Pheophytin Pheophorbide Hydrolase (Pheophytinase=PPH), Plant Cell. 2009, 21(3):767-785,

For heterologous expression of this gene, a synthetic PPH gene (Figure 12) with codons optimized for expression in Trichoderma reesei was synthesized. The N-terminal transit peptide was replaced by a kexin linker (shown in bold in Figure 12), an octapeptide TSVAVEKR. The nucleotide sequence (SEQ ID NO:3) and amino acid sequence (SEQ ID NO:4) of the synthetic PPH gene and polypeptide are shown in Figure 12.

A fusion construct was made consisting of the cellobiohydrolase 1 (cbhl) catalytic core fused to the mature pheophytinase protein via the kexin linker. The expression construct shown in Figure 13, pSMMTrex4-kexPPH, was made by restriction digestion of the synthetic PPH gene using restriction enzymes Spel Sc Ascl and isolating this fragment to be used as insert. The Spel/Ascl cut and alkaline phosphatase-treated Trichoderma vector Trex4, was used in a ligation reaction. The final expression cassette pSMMTrex4-kexPPH contains the promoter and terminator regions of the T reesei cellobiohydrolase 1 (cbhl) gene flanking the PPH synthetic gene, at the 5' and 3' end, respectively. It also contains the Aspergillus nidulans acetamidase, amdS gene as selectable marker for transformation of T reesei

Two other expression constructs were made by fusing the synthetic gene encoding the mature PPH protein directly to two different signal peptides, namely the cellobiohydrolase 1 (cbhl) signal peptide and the Aspergillus tubigensis lipase 3 prepro-signal sequence. The strain used for transformation is Trichoderma reesei, a derivative of the non-GMM strain

RL-P37 from which the genes encoding the two secreted cellobiohydrolases, CBHI and CBHII, and two of the secreted endoglucanases, EGI and EGII₅ have been deleted.

Transformation of T. reesei quad delete Host Strain

The expression construct, pSMMTrex4-kexPPH, containing the A. thaliana pheophytinase gene was transformed into a T reesei by biolistic transformation using the PDS-1000 Helium system (BioRad Cat No. 165-02257).

A suspension of spoies from a quad deleted strain of T,. reesei was prepared. 200μl of spore suspension was spread onto the center of the minimal medium (MM) acetamide plates- MM acetamide plates had the following composition: 0,6g/l acetamide; 1.68g/l CsCl; 20g/l glucose; 20g/I KH₂PO₄, 0.6g/l CaCl₂ 2H₂O; lml/1 100Ox trace elements solution; 20g/l Noble agar, and pH5.5. 100Ox trace elements solution contained 5.0g/l FeSO₄ 7H₂O; 1.6g/l MnSO^ L4g/1 ZnSO₄ 7H₂O and LOg/1 CoCl₂ 6H₂O. The spore suspension was allowed to dry on the surface of MM acetamide medium for 1 hour in the sterile hood. Transformation followed the manufacturer's instructionn 60mg of tungsten particles were placed in a microfuge tube. ImI of ethanol was added and allowed to stand for 15 seconds. The ethanol was removed and the particles were washed three times with sterile dH₂O before 250μl of 50% (v/v) sterile glycerol was added, 25μl of tungsten particle suspension was placed onto a microfuge tube. While continuously vortexing, the following were added: 5μl (100-200 ng/μl) of plasmid DNA, 25μl of 2-5M CaCl₂ and lOμl of 0.1M spermidine. The particles were centrifuged for 3 seconds. The supernatant was removed and the particles were washed with 200μl of 100% ethanol and centrifuged for 3 seconds. The supernatant was removed. 24μl 100% ethanol was added and mixed by pipetting, then 8 μl aliquots of particles were removed and placed in the center of microcarrier disks that were held in a desiccator. Once the tungsten/DNA solution had dried the microcarrier disk was placed in the bombardment chamber along with the plate of MM acetamide with spores and the bombardment process was carried out according to the manufacturer's instructions. After bombardment of the plated spores with the tungsten DNA particles, the plates were incubated at 28⁰C, Transformed colonies were transferred to fresh plates of MM acetamide medium and incubated at 28⁰C. Growth of transformants

After 5 days of growth on MM acetamide plates, transforraants obtained by biolistic transformation and displaying stable morphology were inoculated into 15 ml NREL- Trichoderma Glucose/Sophoiose Defined media in a 50 ml shake flask, NREL-Trich Glue/Soph Defined medium (per liter) consists of (NH₄)₂SO₄ 5g, PIPPS buffer 33 g, Casamino Acids 9g, KH₂PO4 4.5 g, CaCl₂ (Anhydrous) Ig, MgSO₄JH₂O Ig₅ pH to 5,50 adjusted with 50% NaOH with milli-Q H2O bring to 966,5 mL. After sterilization, the following were added: Mazu 5 mL, Glucose/Sophrose 60% 26 mL and 400X T. reesei Trace Metals 2.5 mL, The shake flask cultures were incubated with shaking at 28C for 5 days.

Screening for Recombinant PPH expression

Mycelium was removed by centrifugation and the supernatant was analysed for the presence of the recombinant PPH. The protein profile of the stable transformants was determined by SDS-PAGE using NuPAGE 4-12% and MES as running buffer. Samples of the supernatant were mixed with appropriate volume of 2x sample loading buffer with reducing agent. The gels were stained with Simply blue Safe stain (Invitrogen), Figure 14 shows an SDS gel showing bands representing high levels of PPH protein expressed by transformants, within the molecular weight range of around 50-60 kDa. The strongly stained bands represents the 2 proteins, fusion partner cbhl catalytic core & the lower band represents the PPH protein.

Presence of other putative PPH proteins with high sequence identity to the Arabidopsis thaliana pheophytinase.

Experiments were conducted to identify genes encoding enzymes with pheophytinase/pyropheophytinase activity in the sequence databases and identify signature motifs that could be used to diagnostically identify putative PPHs, The protein sequence of the functionally characterized pheophytinase, namely the chloroplast localized protein from Arabidopsis thaliana described by Schelbert, et al. 2009, Plant Cell 21 (3):767~85, was used as query in BLAST analysis on the non-redundant (nr) protein database of the National Center for Biotechnology Information (NCBI). A number of putative PPH genes were found to be present in different plants of the genus Populus, Vitis, Ricinus, Oryzae sp., Nicotiana, Zect sp, and Physcomitrella. Protein sequences from various species are identified as putative PPHs based on high amino acid sequence identity to the known Arabidopsis PPH. Sequence identity of the different putative PPH to the Arabidopsis PPH ranges from 50 to 63%, as shown in the table below:

A Oryzae Oryzae Zea Physco- Nicotiana Vitis Popuius Ricinus thaliana Sativa sativa j2 Mays mitrella Tabacum Vinifera Trichocarpa Communis pheophy Os] Osj2 Zm Pp Ni Vv Pt Rc -tinaεe AT

AT 56 58 56 50 56 61 63 62

Osj 97 79 48 56 61 58 57

Os]2 78 50 56 61 58 57

Zm 50 55 60 58 58

Pp 48 50 50 51

Nt 67 60 61

Vv 70 66 p_T 66

Rc

Sequence identity as high as 78%, was observed between the putative maize & rice PPHs. Identified PPHs were derived from the following species, followed by NCBI database accession numbers and SEQ ID and Figure no.s: Popuius trichocarpa (XP_Q02314066, SEQ ID NO:5, Fig, 15); Vitis vinifera (XPJ)02271167, SEQ ID NO:6, Fig. 16); Ricinus communis (EEF48653, SEQ ID NO:7, Fig. 17); Ory∑a sativa (japonica cultivar-group) (NP_001057593, SEQ ID NO:8, Fig. 18); Zea mays (NPJ)Ol 141976, SEQ ID NO:9, Fig. 19); Nicotiana tabacum (CAO99125, SEQ ID NOiIO₁ Fig. 20); Oryza sativa Japonica Group (BAG91172, SEQ ID NO: 11, Fig. 21); Physcomitrella patens subsp. patens (XP_00176I725, SEQ ID NO:12, Fig. 22a).

Figure 23 shows an alignment of the functionally characterized Pheophytin Pheophorbide Hydrolase from Arabidopsis thaliana with putative pheophytinases/pyropheophytinases showing several blocks of conserved amino acid residues. The conserved blocks have the following amino acid sequences: LPGFGVG (SEQ ID NO: 13), DFLGQG (SEQ ID NO: 14), GNSLGG (SEQ ID NO: 15), LVKGVTLLNATPFW (SEQ ID NO: 16), HPAA (SEQ ID NO: 17), EDPW (SEQ ID NO: 18), and SPAGHCPH (SEQ ID NO: 19), These conserved blocks can be used to identify new members of pheophytinase/pyropheophytinase family either by searching sequenced genome databases, screening metagenomic libraries or by using these amino acids as degenerate oligonucleotide probes in a PCR to identify new PPH genes present in different chlorophyll containing organisms such as plants, algae, cyanobacteria and photosynthetic chlorophyll containing bacteria.

These conserved PPH motifs can be used individually to search sequences from genome databases, the microbiome metagenome database (JGI-DOE₁ USA) for PPHs. A second conserved motif can be used in addition to the first conserved motif in searching for new PPHs- The GNSLGG (SEQ ID NO: 15) motif contains the active site serine residue which can be used together with the other 6 motifs to identify new PPH candidates from plants, algae and bacteria.

Example 2

Pheophytinase (e.g. a PPH as prepared in example 1 having pyropheophytinase activity) is tested in crude rape seed oil according to the recipe in table 2:

Table 2

Crude rape seed oil is heated to 30⁰C with agitation. Citric acid is added to the oil and the sample is homogenized with a high shear mixer for 20 secondSn After 10 minutes agitation NaOH and water is added. Pheophytinase is then added. The sample is homogenized again with a high shear mixer for 20 seconds. Agitation is continued for 2 hours.

The sample is then heated in a boiling water bath for 10 minutes and then centrifuged at 3000 rcf. for 3 minutes. The oil phase is isolated and remaining water in the oil is removed by vacuum distillation at 3O⁰C and 20 hPa. 75 gram dried oil is heated to 90⁰C and 0,75 gram Silica, Trisyl 300 is added. The oil is agitated with silica for 60 minutes at 9O⁰C. The silica is then separated from the oil by centrifugation or filtration.

In the enzymatic treatment of crude rape seed oil more than 90% of the pheophytin and pyropheophytin in the oil is hydrolysed during formation of phytol and pheophorbide/pyropheophorbide respectively. Silica treatment of the oil after enzyme treatment and drying removes 90% of both pheophorbide and pyropheophorbide in the oil.

Example 3

Pheophytinase is tested in crude rape seed oil according to the recipe in table 3.

Table 3

Crude rape seed oil is heated to 3O⁰C with agitation and water is added. Pheophytinase immobilized on silica is then added. The sample is agitated for 2 hours at 30⁰C and the enzyme is then separated from the oil by centrifugation. By HPLC analysis it is measured that 90% of pheophytin and pyropheophytin are hydrolysed to phytol and pheophorbide/pyropheophorbide respectively .

The oil phase already treated with immobilized pheophytinase is added to 1 gram silica, Trisyl 300. The oil is agitated with silica for 60 minutes at 9O⁰C. The silica is then separated from the oil by centrifugation or filtration.

Silica treatment of the oil after enzyme treatment and drying removes 90% of both pheophorbide and pyropheophorbide. The oil is then deodorized at 240°C and 0.5 hPa for 1 hour. After deodorization 99% of the pheophorbide and pyropheophorbide produced by the enzyme reaction is removed.

Example 4

Pheophytinase is tested in crude rape seed oil according to the recipe in table 4. Table 4

* Lipid acyltransferase activity may be determined as described in WO 2004/064987,

Crude rape seed oil is heated to 55°C with agitation. Citric acid is added to the oil and the sample is homogenized with a high shear mixer for 20 seconds. After 10 minutes agitation NaOH and water is added, Acyltransferase is then added. The sample is homogenized again with a high shear mixer for 20 second. Agitation is continued for 1 hour.

The samples are then centrifuged at 3000 rcf. for 3 minutes. The oil phase is isolated and remaining water in the oil is removed by vacuum distillation at 60⁰C and 20 hPa. 75 gram dried oil is cooled to 3O⁰C and incubated with immobilized pheophytinase for 1 hr. during agitation. The immobilized enzyme is then removed. The oil is heated to 9O⁰C and 0.75 gram silica, Trisyl 300 is added. The oil is agitated with silica for 30 minutes at 90°C The silica is then separated from the oil by centrifugation or filtration. The oil is then deodorized at 25O⁰C and 0.5 hPA for 60 minutes,

In the enzymatic treatment of crude rape seed oil with pheophytinase more than 95% of the pheophytin and pyropheophytm in the oil is hydrolysed during formation of phytol and pheophorbide/pyropheophorbide respectively. Silica treatment of the oil after enzyme treatment removes 90% of both pheophorbide and pyropheophorbide in the oil. The deodorization process removes 95% of the remaining pheophorbide/pyropheophorbide.

Example 5

In this example, the effect of a deodorization step in the refining process on pheophytin, pheophorbide and pyropheophorbide levels in oil is demonstrated. Deodorization is normally the last step in oil refining. Pheophytin, pheophorbide and pyropheophorbide were tested in a model system of refined rape seed oil where these components were added in known concentrations.

Pheophoibide and pyro-pheophorbide were added to refined rape seed oil at a concentration of 2 mg/kg oil (2 ppm) and pheophytin was added as reference at a concentration of 3 ppm (Table 5),

Table 5

1 2 3 4

Refined rape seed oil g 600 600 600 600

Acetone ml 0.600

Pheophorbide, 2 mg/ml ml 0,600

Pyropheophorbide, 2 mg/ml in acetone ml 0.600

Pheophytin, 0,98 rag/ml in

Acetone ml 1.840

Fluorescence detection of pheophytin, pheophorbide and pyropheophorbide

Solutions 2, 3 and 4 from table 5 were diluted with refined rape seed oil to give concentrations of pheophorbide and pyropheophorbide between 3 to 0.04 ppm and 2 to 0,03 ppm respectively. 200 μl of these samples were transferred to a fluorescence microtiter plate and the fluorescence RFU was measured at 25^DC with excitation of 410 nm emission of 672 nm with results shown in Table 6.

Table 6

Pheophytin Pheophorbide Pyropheophorbide mg/ml RFU mg/kg RFU mg/kg RFU

3.005 5048 2.000 9375 2.000 9956

1.494 2613 1.005 4986 0.996 5707

0.757 1461 0.502 2766 0.501 2929

0,468 935 0.309 1927 0.294 2085

0.307 634 0.198 1190 0.213 1495

0.156 382 0.107 687 0.110 846

0.081 222 0.052 394 0.056 449

0.037 146 0.028 251 0,026 251

Based on the results in table 6 calibration curves were constructed and used to calculate unknown samples. 50Og refined iapeseed oil comprising the components defined in Table 5 was deodorized in a round bottom glass flask at 24O⁰C and O.lSrnBar with steam injection for one hour.

Oil samples spiked with pheophytin, pheophorbide and pyropheophorbide (Table 5) were deodorized and samples of the oils before and after deodorization were measured on an Fluorescence microliter plate reader. Based on the calibration curve obtained from standard mixtures of pheophytin, pheophorbide and pyropheophorbide in oil (Table 6) the amount of these components were determined with results shown in Table 7.

Table 7 Fluorescence analysis of oils spiked with pheophytin, pheophorbide and pyropheophorbide before and after deodorization

Pheophorbide Pyropheophorbide Pheophytin

The results in table 3 clearly indicate that the deodorization process reduces the level of pheophytin, pheophorbide and pyropheophorbide in the oil.

The samples were also analysed by HPLC and the chromatograras of the HPLC analysis is shown in Figures 2 to 9. The HPLC chromatograms clearly illustrated the effect of deodorization on the level of pheophytin, pheophorbide and pyropheophorbide. For both, pheophorbide and pyropheophorbide the level after deodorization is below the detection level for these components, and it is therefore not possible to calculate the amount of pheophorbide and pyropheophorbide. Deodorization of oil with pheophytin also removes this component to a level below detection limit, but a new component appears in the sample (fig. 9) which is expected to be pyropheophytin.

Example 6 In this example it is demonstrated that pheophoibide and pyropheophorbide can be removed from oil in the method of the present invention by adsorption on silica, Silica is used in oil processing to remove polar components including phospholipids, soaps and metal ions, but silica is inefficient in removing chlorophyll, pheophytin and pyropheophytin,

10 gram oil (Table 5) was scaled in a Wheaton glass and OJg Trisy] 300 silica was added. The sample is agitated for 10 minutes. Silica is then removed by centrifugation at 4000 rcf for

5 minutes,

5 gram of the oil phase from centrifugation is scaled in a wheaton glass and 0.050 g Trisyl 300 is added. The sample is agitated for 10 minutes. Silica is then removed by centrifugation at 4000 rcf for 5 minutes.

The silica treatment is conducted at two temperatures 25°C and 90°C.

Oil samples spiked with pheophytin, pheophorbide and pyropheophorbide (Table 5) were also treated with silica (Trisyl 300). hi this experiment the effect of temperature was also investigated. The silica treatment was conducted according to the procedure and the samples before silica treatment and after one and two treatments were analyzed by fluorescence, and the amount of the components were calculated based on calibration curves from standard mixtures of the components (Table 6),

The results from the silica treatment are shown in Table 8,

Table 8

Silica Temperature Pheophorbide Pyropheophorbide Pheophytin

Treatment ⁰C RPU ppm ppm Ppm

Sample 1 0 25 82 -0.01 -0.01 -0.01

Sample 1 1 25 68 -0.02 -0.02 -0.02

Sample 1 2 25 78 -0.01 -0,01 -0.01

Sample 1 1 90 59 -0.02 -0.02 -0.02

Sample 1 2 90 52 -0.02 -0.02 -0.02

Sample 2 0 25 9226 1.97

Sample 2 1 25 7410 1.53

Sample 2 2 25 7012 1.44

Sample 2 1 90 3606 0.68

Sample 2 2 90 659 0.10

Sample 3 0 25 10738 2.20

Sample 3 1 25 9090 1.78

Sample 3 2 25 8535 1.64

Sample 3 1 90 5804 1.03 Sample 3 2 90 11 11 0.16

Sample 4 0 25 5164 3.09

Sample 4 1 25 4774 2.83

Sample 4 2 25 5210 3.12

Sample 4 1 90 4669 2.76

Sample 4 2 90 3801 2.21

The results in table 8 confirm that the temperature is very important for the adsorption of pheophorbide and pyropheophorbide on silica. In particular, silica treatment at 9O⁰C is more efficient at removing pheophorbide and pyropheophorbide than treatment at 25°C. The results clearly confirm that two-step silica treatment is much more efficient than a single silica treatment. The results also indicate that pheophytin is not so efficiently adsorbed onto silica. Based on the results it is concluded that the polar degradation products pheophorbide and pyropheophorbide are more easily adsorbed on silica than pheophytin.

These results suggest the use a combination of silica adsorption and deodorization for removal of pheophorbide and pyropheophorbide during oil refining.

Example 7

Cloning and expression of a chlorophyllase from Triticum aestivum (wheat) in Bacillus subtilis

A nucleotide sequence (SEQ ID No. 22) encoding a wheat chlorophyllase (SEQ. ID No. 21, hereinafter wheat chlase) was expressed in Bacillus subtilis with the signal peptide of a B, subtilis alkaline protease (aprE) (see Fig. 31), For optimal expression in Bacillus, a codon optimized gene construct (TRI_CHL) was ordered at GenScript (GenScript Corporation, Piscataway, NJ 08854, USA).

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

The construct TRI_CHL was digested with BssHII and Pad and ligated with T4 DNA ligase into BssHII and Pad digested pBNppt.

The ligation mixture was transformed into E coli TOPlO cells. The sequence of the BssHII and Pac insert containing the TRI_CHL gene was confirmed by DNA sequencing (DNA Technology AJS, Risskov, Denmark) and one of the correct plasrnid clones was designated pBN-TRI_CHL (Figure 32). pBN-TRI_CHL was transformed into B subtilis strain BG 6002 a derivative of AK 220O₅ as described in WO 2003/099843.

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

Example 8

Cloning and expression of a chlorophyllase from Chlamydomonas reinhardtii (green algae) in Bacillus subtilis

A nucleotide sequence (SEQ ID No. 24) encoding a Chlamydomonas chlorophyllase (SEQ. ID No. 23, hereinafter chlamy chlase) was expressed in Bacillus subtilis with the signal peptide of a B, subtilis alkaline protease (aprE) (see Fig.s 33 and 34). For optimal expression in Bacillus, a codon optimized gene construct (CHL-CHL) was ordered at GenScript (GenScript Corporation, Piscataway, NJ 08854, USA).

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

The construct CHL_CHL was digested with BssHII and Pad and ligated with T4 DNA ligase into BssHII and Pad digested pBNppt

The ligation mixture was transformed into E. coli TOPlO cells. The sequence of the BssHII and Pac insert containing the CHL_CHL gene was confirmed by DNA sequencing (DNA Technology A/S₅ Risskov, Denmark) and one of the correct plasmid clones was designated pBN-CHL_^CHL (Figure 20). pBN-CHL_CHL was transformed into B subtilis strain BG 6002 a derivative of AK 2200, as described in WO 2003/099843.

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

Example 9 Cloning and expression of an N-terminal truncated variant of Triticum aestivum chlorophyllase

In this example, a variant of Triticum aestivum chlorophyllase was constructed lacking the N- terminal 16 amino acids compared to the wild-type enzyme. This variant is designated Triticum NdI-16 and its amino acid sequence is shown in SEQ ID NO:25. The nucleotide sequence (SEQ ID NO:26) encoding the variant was expressed in Bacillus subtilis with the signal peptide of a B. subtilis alkaline protease (aprE). For optimal expression in Bacillus, a codon optimized gene construct (TRI_CHL-S) was ordered at GenScript (GenScript Corporation, Piscataway, NJ 08854, USA).

The construct TRI_CHL-S contains 20 nucleotides with a BssHII restriction site upstream to the wheat chlase variant coding region to allow fusion to the aprE signal sequence and a Pad restriction site following the coding region for cloning into the bacillus expression vector pBNppt.

The construct TRI_CHL-S was digested with BssHII and Pad and Hgated with T4 DNA ligase into BssHII and Pad digested pBNppt

The ligation mixture was transformed into E. coli TOPlO cells. The sequence of the BssHII and Pac insert containing the TRI_CHL gene was confirmed by DNA sequencing (DNA Technology A/S, Risskov, Denmark) and one of the correct plasmid clones was designated pBN-TRLCHL-S. pBN-TRI_CHL-S was transformed into B.subtilis strain BG 6002 a deiivative of AK 2200 (See US2003/015859 and WO/2003/099843).

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

Example 10

Pyropheophytinase activity of chiorophyllases

The pyropheophytinase and pheophytinase activity of the enzymes described in Examples 7 to 9 was determined, and compared to the activity of Arabidopsis thaliana chlorophyllase (SEQ ID NO:20). The results are shown in the following table:

Pheophytinase or pyropheophytinase activity as shown in the above table refers to the number of micromoles of substrate (pheophytin or pyropheophytin) hydrolysed per minute in the assay.

The above table shows the activity of various chlorophyll ases on pheophytin and pyropheophytin, and the activity ratio is calculated as pheophytinase activity/pyropheophytinase activity. Lowering the activity ratio indicates a shift towards increased ability to hydrolyze pyropheophytin which is an advantage in oils with significant levels of pyropheophytin.

The Arabidopsis chlorophyll ase has a high ratio of pheophytinase to pyropheophytinase activity. However, surprisingly the enzymes from Chlamydomonas and Triticum have a much lower activity ratio with relatively increased activity on pyropheophytin. Furthermore, truncation of the N-terminus of chlorophyllases can generate improved variants with a lower activity ratio compared to the full length enzyme, as demonstrated for a variant of the Triticum chlorophyllase lacking the N-terrninal 16 amino acids compared to the wild-type enzyme . 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 method for treating a pyropheophytin-containing composition, comprising contacting the composition with an enzyme which is capable of hydrolysing pyropheophytin.

2,. A method according to claim 1, wherein the composition comprises a plant-derived oil.

3. A method according to claim 1 or claim 2, wherein the enzyme comprises a pheophytinase or pheophytin pheophorbide hydrolasen

4. A method according to any preceding claim, wherein the enzyme is derived from Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera, Oiγzo sativa, Zea mays,

Nicotiana tabacum, Ostreococcus lucimarhms, Ostreococcus taurii, Physcomitrella patens, Phaeodactylum tricornutum, Chlamydomonas reinhardtii, Triticum aestivum or Micromonas sp. RCC299.

5. A method according to any preceding claim, wherein, the enzyme comprises an amino acid sequence selected from: LPGFGVG (SEQ ID NO:13), DFLGQG (SEQ ID NO: 14),

GNSLGG (SEQ ID NO: 15), LVKGVTLLNATPFW (SEQ ED NO: 16), HPAA (SEQ ID NO: 17), EDPW (SEQ ID NO: 18), and SPAGHCPH (SEQ ID NO: 19).

6. A method according to any preceding claim, wherein the enzyme comprises a polypeptide sequence as defined in SEQ ID NO:1, any one of SEQ ID NO:s 4 to 12, or any one of SEQ ID NOs:21 , 23 or 25, or a functional fragment or variant thereof

7. A method according to claim 6, wherein the enzyme comprises a polypeptide sequence having at least 75% sequence identity to SEQ ID NO: I₅ any one of SEQ ID NO:s 4 to 12, or any one of SEQ ID NOs:21, 23 or 25, over at least 50 amino acid residues.

8. A method according to any preceding claim, wherein pyropheophytin in the composition is hydrolysed to form pyropheophorbide.

9. A method according to claim 8, further comprising a step of removing pyropheophorbide from the composition.

10. A method according to claim 9, wherein the method comprises a deodorization step.

11. A method according to claim 9 or claim 10, wherein the method comprises a step of silica treatment.

12. A method according to claim 11 , wherein the method comprises a two or more silica treatment steps.

13. A method according to claim 11 or claim 12, wherein the silica treatment is performed at about 70 to 110⁰C.

14, A method according to any preceding claim, wherein the enzyme which is capable of hydrolysing pyropheophytin is immobilized on a solid support.

15- A method according to any preceding claim, further comprising contacting the composition with an enzyme having chlorophyllase activity.

16. A method according to claim 15, wherein the enzyme having chlorophyllase activity is immobilized on a solid support.

17. A method according to any of claims 2 to 16, wheiein the composition comprises an oil selected from rice bran, soy, canola, palm, olive, cottonseed, com, palm kernel, coconut, peanut, sesame or sunflower oil.

18. A method according to any preceding claim, wherein the concentration of pyropheophytin in the composition is reduced by at least 10% compared to the concentration of pyropheophytin present in the composition before treatment.

19. A process for refining a plant oil, comprising a method as defined in any preceding claim.

20. A process according to claim 19, further comprising a hexane extraction and/or degumming step.

21. Use of a polypeptide having pyropheophytinase activity for removing pyropheophytin contamination from a composition.

22. An apparatus for the enzymatic treatment of a pyropheopytin- containing composition, comprising (a) a plant oil refining apparatus; and (b) a polypeptide having pyropheophytinase activity operably integrated into the plant oil refining apparatus, such that the polypeptide is capable of hydrolyzing pyropheophytin in the composition during refining of the composition.

23. A composition comprising a polypeptide having pyropheophytinase activity immobilized on silica.

24. A polypeptide having a sequence as defined in SEQ ID NO:4, or encoded by a sequence as defined in SEQ ID N0:3, or a functional variant or fragment thereof having pyropheophytinase activity.

25. A composition obtainable by the process or method of any of claims 1 to 20,

26. A method according to any of claims 1 to 18, wherein the enzyme has a pheophytinase to pyropheophytinase activity ratio of less than 80.

27. A polypeptide having a sequence as defined in SEQ ID NO:25, or a functional variant or fragment thereof having pyropheophytinase activity.