Compositions and Methods for Enzymatic Decolorization of Chlorophyll

Abstract

The invention provides compositions and methods for the enzymatic treatment (“bleaching” or “de-colorizing”) of chlorophyll-comprising compositions, e.g., algae preparations, chlorophyll-containing or chlorophyll-contaminated feeds, foods or oils, for example, vegetable oils, including oils processed from oilseeds, such as canola (rapeseed) oil or soybean oil, or oil fruits, such as palm oil. In one aspect, the invention provides methods using a chlorophyllase enzyme for the enzymatic hydrolysis of chlorophyll in an algae, an animal (e.g., a fish) or plant preparation, a food or an oil. In one aspect, the chlorophyllase is immobilized onto a silica. The invention also provides compositions of manufacture and detergents.

1 . An isolated, synthetic or recombinant nucleic acid comprising
 (a) a nucleic acid sequence
 (i) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:19, wherein the nucleic acid encodes at least one polypeptide having an esterase or a chlorophyllase activity, or 
 (ii) that hybridizes under stringent conditions to the complement of a nucleic acid comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO: 19, wherein the stringent conditions include a wash step comprising a wash in 0.2×SSC at a temperature of about 65° C. for about 15 minutes, wherein the nucleic acid encodes at least one polypeptide having an esterase or a chlorophyllase activity; 
   (b) the nucleic acid sequence of (a), wherein the sequence identity is over a region of at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or more residues, or the full length of a gene or a transcript;   (c) the nucleic acid sequence of (a), wherein the nucleic acid sequence encodes a polypeptide having a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 or SEQ ID NO:20;   (d) the nucleic acid sequence of any of (a) to (c), wherein the sequence comparison algorithm is a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall-p blastp-d “nr pataa”-F F, and all other options are set to default;   (e) the nucleic acid sequence of any of (a) to (d), wherein the esterase or chlorophyllase activity comprises enzymatic modification of a chlorophyll molecule; or an enzymatic modification comprising catabolism of a chlorophyll molecule; or a chlorophyllase activity; or a chlase activity; or a chlorophyll chlorophyllido-hydrolyase activity;   (f) the nucleic acid sequence of any of (a) to (g), wherein the polypeptide's activity is thermostable, or the polypeptide retains enzymatic activity under conditions comprising a temperature range of between about 37° C. to about 95° C., or between about 55° C. to about 85° C., or between about 70° C. to about 75° C., or between about 70° C. to about 95° C., or between about 90° C. to about 95° C.;   (g) the nucleic acid sequence of any of (a) to (e), wherein the polypeptide's activity is thermotolerant, or the polypeptide retains enzymatic activity after exposure to a temperature in the range from greater than 37° C. to about 95° C., from greater than 55° C. to about 85° C., or between about 70° C. to about 75° C., or from greater than 90° C. to about 95° C.;   (h) the nucleic acid sequence of any of (a) to (g), wherein the polypeptide retains activity under conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH 11 or more basic, or, the polypeptide retains activity under conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4 or more acidic;   (i) the nucleic acid sequence of any of (a) to (h), wherein the polypeptide has a specific activity from about 100 to about 1000 units per milligram of protein, or, from about 500 to about 750 units per milligram of protein;   (j) the nucleic acid sequence of any of (a) to (i), encoding a polypeptide lacking a signal sequence;   (k) the nucleic acid sequence of any of (a) to (j), further comprising a heterologous nucleic acid sequence or a heterologous nucleic acid sequence encoding a heterologous peptide or polypeptide:   (l) the nucleic acid sequence of any of (j), wherein the heterologous nucleic acid sequence comprises a nucleic acid sequence encoding a heterologous signal sequence (signal peptide), a heterologous prepro domain, a heterologous chlorophyllase signal sequence (signal peptide), a heterologous non-chlorophyllase signal sequence (signal peptide), an immunogenic fragment, an antibody binding peptide, an epitope, a motif, a binding site, a substrate binding site or an enzymatic cleavage site;   (m) the nucleic acid sequence of (k) or (l), wherein the heterologous polypeptide or peptide is amino terminal to, carboxy terminal to or on both ends of a signal peptide (SP), prepro domain or catalytic domain (CD); or   (n) a nucleic acid sequence completely complementary to the nucleic acid sequence of any of (a) to (m).   
     
     
         2 - 20 . (canceled) 
     
     
         21 . A nucleic acid probe for identifying a nucleic acid encoding a polypeptide having a chlorophyllase activity or involved in the catabolism of a chlorophyll molecule, wherein the probe comprises
 (a) a nucleic acid comprising at least about 10, 15, 20, 25, 30, 35, 40, 45, 50 or more consecutive residues of the nucleic acid sequence of  claim 1 ;   (b) at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more consecutive bases of a sequence comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:19, or   (c) the probe of (a) or (b) comprising an oligonucleotide comprising at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, about 60 to 100, or about 50 to 150 consecutive bases.   
     
     
         22 - 24 . (canceled) 
     
     
         25 . An amplification primer pair for amplifying a nucleic acid encoding a polypeptide having a chlorophyllase activity or involved in the catabolism of a chlorophyll molecule, wherein the amplification primer pair
 (a) is capable of amplifying a nucleic acid comprising the sequence of  claim 1 ;   (b) the amplification primer pair of (a), wherein a member of the pair comprises an oligonucleotide comprising at least about 10 to 50 consecutive bases of the sequence, or, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive bases of the sequence, or   (c) the amplification primer pair of (a) or (b), wherein the pair comprises a first member having a sequence as set forth by about the first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more residues of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:19, and a second member having a sequence as set forth by about the first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more residues of the complementary strand of the first member.   
     
     
         26 - 27 . (canceled) 
     
     
         28 . A nucleic acid comprising (a) a sequence generated by amplification of a polynucleotide using an amplification primer pair as set forth in  claim 25 , or
 (b) the nucleic acid of (a), wherein the amplification is by polymerase chain reaction (PCR), or, the nucleic acid generated by amplification by polymerase chain reaction (PCR) of a gene library, or,   (c) the nucleic acid of (a) or (b), wherein the nucleic acid is generated by amplification of an environmental gene library.   
     
     
         29 - 32 . (canceled) 
     
     
         33 . A method of amplifying a nucleic acid encoding a polypeptide having a chlorophyllase activity or involved in the catabolism of a chlorophyll molecule, comprising amplification of a template nucleic acid with an amplification primer pair capable of amplifying the nucleic acid sequence of  claim 1 . 
     
     
         34 . An expression cassette, a vector, or a cloning or expression vehicle comprising
 (a) a nucleic acid comprising the nucleic acid sequence of  claim 1 ;   (b) the expression cassette, a vector, or a cloning or expression vehicle of (a), comprising or contained in a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage, an artificial chromosome, a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC); or wherein the viral vector comprises an adenovirus vector, a retroviral vector or an adeno-associated viral vector.   
     
     
         35 - 38 . (canceled) 
     
     
         39 . A transformed cell comprising, or having contained within, (a) the nucleic acid sequence of  claim 1 ; (b) the expression cassette, a vector, or a cloning or expression vehicle of  claim 34 ; or, (c) the cell of (a) or (b), wherein the transformed cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell. 
     
     
         40 - 41 . (canceled) 
     
     
         42 . A transgenic non-human animal comprising, or having contained within, (a) the nucleic acid sequence of  claim 1 ; (b) the expression cassette, a vector, or a cloning or expression vehicle of  claim 34 ; or (c) the transgenic non-human animal of (a) or (b) wherein the animal is a mouse or a rat. 
     
     
         43 . (canceled) 
     
     
         44 . A transgenic plant or transgenic seed comprising, or having contained within, (a) the nucleic acid sequence of  claim 1 ; or (b) the expression cassette, a vector, or a cloning or expression vehicle of  claim 34 ; wherein the transgenic plant or transgenic seed is or is of a corn plant, a sorghum plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed plant, a soybean plant, a rice plant, a barley plant, a grass, or a tobacco plant. 
     
     
         45 - 47 . (canceled) 
     
     
         48 . An antisense oligonucleotide comprising (a) a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to the nucleic acid sequence of  claim 1 ; or (b) the antisense oligonucleotide of (a), wherein the antisense oligonucleotide is between about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, about 60 to 100, or about 75 to 125 or more bases in length. 
     
     
         49 . (canceled) 
     
     
         50 . A method of inhibiting the translation of a message in a cell comprising administering to the cell or expressing in the cell an antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to the nucleic acid sequence of  claim 1 . 
     
     
         51 . A double-stranded inhibitory RNA (RNAi) molecule comprising (a) a subsequence of the nucleic acid sequence of  claim 1 ; or (b) the double-stranded inhibitory RNA (RNAi) molecule of (a), wherein the RNAi is about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length. 
     
     
         52 . (canceled) 
     
     
         53 . A method of inhibiting the expression of an enzyme in a cell comprising administering to the cell or expressing in the cell a double-stranded inhibitory RNA (iRNA), wherein the RNA comprises a subsequence of the nucleic acid sequence of  claim 1 , or the double-stranded inhibitory RNA (RNAi) molecule of  claim 51 . 
     
     
         54 . An isolated, synthetic or recombinant polypeptide
 (a) comprising an amino acid sequence
 (i) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or has 100% (complete) sequence identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 or SEQ ID NO:20, wherein the polypeptide has an esterase or a chlorophyllase activity or comprises an immunogenic fragment capable of eliciting a humoral antibody or cellular immune response specific to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 or SEQ ID NO:20, or, 
 (ii) encoded by the nucleic acid of  claim 1 , wherein the polypeptide has an esterase or a chlorophyllase activity or comprises an immunogenic fragment capable of eliciting a humoral antibody or cellular immune response specific to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 or SEQ ID NO:20, 
 (iii) encoded by the nucleic acid of  claim 28 , wherein the polypeptide has a chlorophyllase activity or is involved in the catabolism of a chlorophyll molecule, 
 (iv) the amino acid sequence of (i) or (ii), wherein the sequence identity is calculated over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75 or 100 residues; 
   (b) the polypeptide of (a), wherein the sequence comparison algorithm is a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall-p blastp-d “nr pataa”-F F, and all other options are set to default;   (c) the polypeptide of (a) or (b), wherein the esterase or chlorophyllase activity comprises enzymatic modification of a chlorophyll molecule; or an enzymatic modification comprising catabolism of a chlorophyll molecule; or a chlorophyllase activity; or a chlase activity; or a chlorophyll chlorophyllido-hydrolyase activity;   (d) the polypeptide of (a) to (c), wherein the polypeptide's activity is thermostable, or the polypeptide retains enzymatic activity under conditions comprising a temperature range of between about 37° C. to about 95° C., or between about 55° C. to about 85° C., or between about 70° C. to about 75° C., or between about 70° C. to about 95° C., or between about 90° C. to about 95° C.;   (e) the polypeptide of (a) to (c), wherein the polypeptide's activity is thermotolerant, or the polypeptide retains enzymatic activity after exposure to a temperature in the range from greater than 37° C. to about 95° C., from greater than 55° C. to about 85° C., or between about 70° C. to about 75° C., or from greater than 90° C. to about 95° C., or wherein the thermotolerance comprises retention of at least half of the specific activity of the enzyme at 37° C. after being heated to an elevated temperature, or the thermotolerance comprises retention of specific activity at 37° C. in the range from about 500 to about 1200 units per milligram of protein after being heated to an elevated temperature;   (f) the polypeptide of any of (a) to (e), wherein the polypeptide retains activity under conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH 11 or more basic, or, the polypeptide retains activity under conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4 or more acidic;   (g) the polypeptide of any of (a) to (f), wherein the polypeptide has a specific activity from about 100 to about 1000 units per milligram of protein, or, from about 500 to about 750 units per milligram of protein; or wherein the enzyme activity comprises a specific activity at about 37° C. in the range from about 100 to about 1000 units per milligram of protein, from about 500 to about 750 units per milligram of protein, from about 500 to about 1200 units per milligram of protein, or from about 750 to about 1000 units per milligram of protein,   (h) the polypeptide of any of (a) to (i) lacking a signal sequence;   (i) the polypeptide of any of (a) to (h) further comprising a heterologous nucleic acid sequence or a heterologous nucleic acid sequence encoding a heterologous peptide or polypeptide;   (j) the polypeptide of any of (i) wherein the heterologous nucleic acid sequence comprises a nucleic acid sequence encoding a heterologous signal sequence (signal peptide), a heterologous prepro domain, a heterologous chlorophyllase signal sequence (signal peptide), a heterologous non-chlorophyllase signal sequence (signal peptide), an immunogenic fragment, an antibody binding peptide, an epitope, a motif, a binding site, a substrate binding site or an enzymatic cleavage site;   (k) the polypeptide of (i) or (j), wherein the heterologous polypeptide or peptide is amino terminal to, carboxy terminal to or on both ends of a signal peptide (SP), prepro domain or catalytic domain (CD);   (l) the polypeptide of any of (a) to (k), wherein the polypeptide comprises an immunogenic fragment capable of eliciting a humoral antibody or cellular immune response;   (m) the polypeptide of any of (a) to (l), wherein the polypeptide comprises at least one glycosylation site, or the polypeptide comprises an N-linked glycosylation, or the polypeptide is glycosylated after being expressed in a  P. pastoris  or a  S. pombe;      (n) the polypeptide of any of (a) to (m), wherein the polypeptide comprises at least one amino acid residue conservative substitution;   (o) the polypeptide of (n), wherein the conservative substitution comprises replacement of an aliphatic amino acid with another aliphatic amino acid; replacement of a Serine with a Threonine or vice versa; replacement of an acidic residue with another acidic residue; replacement of a residue bearing an amide group with another residue bearing an amide group, exchange of a basic residue with another basic residue; or, replacement of an aromatic residue with another aromatic residue, or a combination thereof; or   (p) the polypeptide of (o), wherein the aliphatic residue comprises Alanine, Valine, Leucine, Isoleucine or a synthetic equivalent thereof; the acidic residue comprises Aspartic acid, Glutamic acid or a synthetic equivalent thereof; the residue comprising an amide group comprises Aspartic acid, Glutamic acid or a synthetic equivalent thereof; the basic residue comprises Lysine, Arginine or a synthetic equivalent thereof; the aromatic residue comprises Phenylalanine, Tyrosine or a synthetic equivalent thereof.   
     
     
         55 - 79 . (canceled) 
     
     
         80 . A protein preparation comprising the polypeptide of  claim 54 , wherein the protein preparation comprises a liquid, a solid or a gel. 
     
     
         81 . A heterodimer comprising (a) the polypeptide of  claim 54  and a second domain; (b) the heterodimer of (a) wherein the second domain is a polypeptide and the heterodimer is a fusion protein; or (c) the heterodimer of (a) or (b), wherein the second domain is or comprises an epitope, an immunogen or a tag. 
     
     
         82 - 83 . (canceled) 
     
     
         84 . A homodimer comprising (a) the polypeptide of  claim 54 ; or (b) the homodimer of (a) wherein the homodimer is a fusion protein. 
     
     
         85 . An immobilized polypeptide, wherein the polypeptide comprises (a) the polypeptide of  claim 54 ; or (b) the immobilized polypeptide of (a), wherein the polypeptide is immobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an array or a capillary tube. 
     
     
         86 . (canceled) 
     
     
         87 . An array comprising (a) an immobilized polypeptide comprising the polypeptide of  claim 54 ; (b) an immobilized nucleic acid comprising the nucleic acid of  claim 1 . 
     
     
         88 . (canceled) 
     
     
         89 . An isolated, synthetic or recombinant antibody (a) that specifically binds to the polypeptide of  claim 54 ; or (b) the antibody of (a), wherein the antibody is a monoclonal or a polyclonal antibody. 
     
     
         90 . (canceled) 
     
     
         91 . A hybridoma comprising an antibody that specifically binds to the polypeptide of  claim 54 . 
     
     
         92 . A method of isolating or identifying a polypeptide with enzyme activity comprising:
 (a) providing the antibody of  claim 89 ;   (b) providing a sample comprising polypeptides; and   (c) contacting the sample of step (b) with the antibody of step (a) under conditions wherein the antibody can specifically bind to the polypeptide, thereby isolating or identifying a polypeptide having enzyme activity.   
     
     
         93 - 94 . (canceled) 
     
     
         95 . A method of producing a recombinant polypeptide comprising
 (i): (a) providing a nucleic acid operably linked to a promoter, wherein the nucleic acid comprises the nucleic acid sequence of  claim 1 ; and (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide; or   (ii) the method of (i), further comprising transforming a host cell with the nucleic acid of step (a) followed by expressing the nucleic acid of step (a), thereby producing a recombinant polypeptide in a transformed cell.   
     
     
         96 . (canceled) 
     
     
         97 . A method for identifying a polypeptide having enzymatic activity comprising:
 (a) providing the polypeptide of  claim 54 ;   (b) providing an enzyme substrate; and   (c) contacting the polypeptide with the substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of a reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of the reaction product detects a polypeptide having an enzyme activity.   
     
     
         98 - 100 . (canceled) 
     
     
         101 . A method for identifying a modulator of an enzyme activity comprising:
 (i) (a) providing the polypeptide of as set forth in  claim 54 ;   (b) providing a test compound;   (c) contacting the polypeptide of step (a) with the test compound of step (b) and measuring an activity of the enzyme, wherein a change in the enzyme activity measured in the presence of the test compound compared to the activity in the absence of the test compound provides a determination that the test compound modulates the enzyme activity;   (ii) the method of (i), wherein the enzyme activity is measured by providing an enzyme substrate and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product, or, an increase in the amount of the substrate or a decrease in the amount of a reaction product;   (iii) the method of (ii), wherein a decrease in the amount of the substrate or an increase in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an activator of an enzyme activity; or   (iv) the method of (ii), wherein an increase in the amount of the substrate or a decrease in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an inhibitor of an enzyme activity.   
     
     
         102 - 114 . (canceled) 
     
     
         115 . A method for isolating or recovering a nucleic acid encoding a polypeptide with an enzyme activity from a sample comprising:
 (A)(i) (a) providing the amplification primer pair of  claim 25 ;
 (b) isolating a nucleic acid from the sample or treating the sample such that nucleic acid in the sample is accessible for hybridization to the amplification primer pair; and, 
 (c) combining the nucleic acid of step (b) with the amplification primer pair of step (a) and amplifying nucleic acid from the sample, thereby isolating or recovering a nucleic acid encoding a polypeptide with an enzyme activity from an sample; 
   (ii) the method of (i), wherein the sample is or comprises an environmental sample;   (iii) the method of (ii), wherein the environmental sample comprises a water sample, a liquid sample, a soil sample, an air sample or a biological sample; or   (iv) the method of (iii), wherein the biological sample is derived from a bacterial cell, a protozoan cell, an insect cell, a yeast cell, a plant cell, a fungal cell or a mammalian cell; or   (B) (i) (a) providing a polynucleotide probe comprising the sequence of  claim 1 ;   (b) isolating a nucleic acid from the sample or treating the sample such that nucleic acid in the sample is accessible for hybridization to a polynucleotide probe of step (a);   (c) combining the isolated nucleic acid or the treated sample of step (b) with the polynucleotide probe of step (a); and   (d) isolating a nucleic acid that specifically hybridizes with the polynucleotide probe of step (a), thereby isolating or recovering a nucleic acid encoding a polypeptide with an enzyme activity from the sample;   (ii) the method of (i), wherein the sample is or comprises an environmental sample;   (iii) the method of (ii), wherein the environmental sample comprises a water sample, a liquid sample, a soil sample, an air sample or a biological sample; or   (iv) the method of (iii), wherein the biological sample is derived from a bacterial cell, a protozoan cell, an insect cell, a yeast cell, a plant cell, a fungal cell or a mammalian cell.   
     
     
         116 - 119 . (canceled) 
     
     
         120 . A method of generating a variant of a nucleic acid encoding a polypeptide with an enzyme activity comprising:
 (i) (a) providing a template nucleic acid comprising the sequence of  claim 1 ; and   (b) modifying, deleting or adding one or more nucleotides in the template sequence, or a combination thereof, to generate a variant of the template nucleic acid;   (ii) the method of (i), further comprising expressing the variant nucleic acid to generate a variant polypeptide;   (iii) the method of (i) or (ii), wherein the modifications, additions or deletions are introduced by a method comprising error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR) and a combination thereof;   (iv) the method of (i) or (ii), wherein the modifications, additions or deletions are introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof;   (v) the method of any of (i) to (iv), wherein the method is iteratively repeated until a variant polypeptide having an altered or different activity or an altered or different stability or an altered structure from that of a polypeptide encoded by the template nucleic acid is produced;   (vi) the method of (v), wherein the variant polypeptide is thermotolerant, and retains some activity after being exposed to an elevated temperature; or, the variant polypeptide has increased glycosylation as compared to the enzyme encoded by a template nucleic acid; or, the variant polypeptide has activity under a high temperature, wherein the polypeptide encoded by the template nucleic acid is not active under the high temperature;   (vii) the method of any of (i) to (iv), wherein the method is iteratively repeated until a polypeptide coding sequence having an altered codon usage from that of the template nucleic acid is produced; or   (viii) the method of any of (vii), wherein the method is iteratively repeated until a gene having higher or lower level of message expression or stability from that of the template nucleic acid is produced.   
     
     
         121 - 129 . (canceled) 
     
     
         130 . A method for modifying codons in a nucleic acid encoding a polypeptide with enzymatic activity to increase or decrease its expression in a host cell comprising:
 (i) (a) providing a nucleic acid encoding a polypeptide with an enzyme activity comprising the sequence of  claim 1 ; and,   (b) identifying a non-preferred or a less preferred codon in the nucleic acid of step (a) and replacing it with a preferred or neutrally used codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to increase its expression in a host cell; or,   (ii) (a) providing a nucleic acid encoding a polypeptide with an enzyme activity comprising the sequence of  claim 1 ; and,   (b) identifying a codon in the nucleic acid of step (a) and replacing it with a different codon encoding the same amino acid as the replaced codon, thereby modifying codons in a nucleic acid encoding an enzyme; or   (iii) (a) providing a nucleic acid encoding a polypeptide comprising the sequence of  claim 1 ; and   (b) identifying at least one preferred codon in the nucleic acid of step (a) and replacing it with a non-preferred or less preferred codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in a host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to decrease its expression in a host cell; or   (iv) the method of any of (i) to (iii), wherein the host cell is a bacterial cell, a fungal cell, an insect cell, a yeast cell, a plant cell or a mammalian cell.   
     
     
         131 - 149 . (canceled) 
     
     
         150 . An isolated, synthetic or recombinant signal sequence consisting of a sequence as set forth in residues 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 98, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1 to 43 or 1 to 44, of (a) SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 or SEQ ID NO:20; or, (b) the polypeptide sequence of  claim 54 . 
     
     
         151 . A chimeric polypeptide comprising (a) at least a first domain comprising signal peptide (SP) having the sequence of as set forth in  claim 150 , and at least a second domain comprising a heterologous polypeptide or peptide, wherein the heterologous polypeptide or peptide is not naturally associated with the signal peptide (SP), or
 (b) the chimeric polypeptide of (a), wherein the heterologous polypeptide or peptide is not an esterase or chlorophyllase or is not involved in the enzymatic modification of a chlorophyll molecule or   (c) the chimeric polypeptide of (a) or (b), wherein the heterologous polypeptide or peptide is amino terminal to, carboxy terminal to or on both ends of the signal peptide (SP) or a catalytic domain (CD).   
     
     
         152 - 153 . (canceled) 
     
     
         154 . An isolated, synthetic or recombinant nucleic acid encoding a chimeric polypeptide, wherein the chimeric polypeptide comprises at least a first domain comprising signal peptide (SP) having a sequence as set forth in  claim 150  and at least a second domain comprising a heterologous polypeptide or peptide, wherein the heterologous polypeptide or peptide is not naturally associated with the SP. 
     
     
         155 - 168 . (canceled) 
     
     
         169 . A method or industrial process comprising:
 (A) enzymatic treatment of chlorophyll-containing or chlorophyll-contaminated compositions comprising:   (i) (a) providing a chlorophyll-containing or chlorophyll-contaminated composition;   (b) providing at least one polypeptide having a chlorophyllase activity or a chlorophyll catabolic enzyme activity; and   (c) reacting the composition of step (a) with the polypeptide of step (b) under conditions wherein the polypeptide can catalyze a chlorophyll-modifying reaction;   (ii) the method of (i), wherein the polypeptide has the amino acid sequence of  claim 54 ;   (iii) the method of (i) or (ii), wherein the chlorophyll-containing or chlorophyll-contaminated composition comprises a food, an oil or a feed or composition derived from a plant, an animal or an algae, or a mixture thereof;   (iv) the method of any of (i) to (iii), wherein the polypeptide is an enzyme or a catalytic antibody;   (v) the method of (iv), wherein the polypeptide is immobilized, or the polypeptide is immobilized on an inorganic support or organic support, or a support comprising alumina, celite, Dowex-1-chloride, glass beads, silica gel, alginate hydrogel or alginate bead or equivalent, or a support comprising an inorganic support or organic support, or a support comprising alumina, celite, Dowex-1-chloride, glass beads, silica gel, alginate hydrogel or alginate bead or equivalent, or a support comprising an inorganic support or organic support comprising alumina, celite, Dowex-1-chloride, glass beads, silica gel, alginate hydrogel or alginate bead or equivalent;   (vi) the method of any of (i) to (v), wherein at least one step of the method is performed in a reaction vessel; or at least one step is performed in a reaction vessel comprising a gravitational gum separation device or a holding tank; or at least one step is performed in a cell extract; or at least one step is performed in a whole cell;   (vii) the method of any of (i) to (vi), wherein the chlorophyll-containing or chlorophyll-contaminated composition comprises a plant material, plant oil or plant extract, or the plant material, plant oil or plant extract comprises a vegetable oil or a seed oil, or the vegetable oil comprises a palm oil or a canola oil, or the plant material, plant oil or plant extract comprises an algae preparation;   (viii) the method of any of (i) to (vii), wherein the method further comprises use of a lipoxygenase;   (ix) the method of any of (i) to (viii), wherein the chlorophyllase activity comprises catalyzing the cleavage of a tetrapyrrole macrocyclic ring and oxygenolytic cleavage of the macrocyclic ring;   (x) the method of any of (i) to (ix), wherein the method further comprises removal of a chlorophilide generated by enzymatic degradation of a chlorophyll by adsorbing onto a silica gel or equivalent;   (xi) the method of any of (i) to (x), wherein the chlorophyll-containing or chlorophyll-contaminated composition comprises a textile or fabric, or the chlorophyll-containing or chlorophyll-contaminated composition comprises a wood or paper product or by-product, or the wood or paper product or by-product comprises a wood pulp or paper pulp, or the chlorophyll-containing or chlorophyll-contaminated composition comprises a non-wood paper product or by-product, or the non-wood or paper product comprises a rice paper;   (xii) the method of any of (i) to (xi), wherein the method further comprises a caustic neutralization step;   (xiii) the method of any of (i) to (xii), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a chlorophilide generated by the enzymatic degradation of the chlorophyll;   (xiv) the method of any of (i) to (xiii), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a pheophorbide generated by the enzymatic degradation of the pheophytin; or   (xv) the method of any of (i) to (xiv), wherein the method further comprises addition of a phospholipase; or the method further comprises addition of a phospholipase C;   (B) enzymatic treatment of chlorophyll-containing or chlorophyll-contaminated compositions comprising:   (i) (a) providing a chlorophyll-containing or chlorophyll-contaminated composition;   (b) providing at least one polypeptide having a chlorophyllase activity or a chlorophyll catabolic enzyme activity; and   (c) reacting the composition of step (a) with the polypeptide of step (b) under conditions wherein the polypeptide can catalyze a chlorophyll-modifying reaction;   (ii) the industrial process of (i), wherein the polypeptide has the sequence of  claim 54 ;   (iii) the industrial process of (i) or (ii), wherein the chlorophyll-containing or chlorophyll-contaminated composition comprises a food, an oil or a feed or composition derived from a plant, an animal or an algae, or a mixture thereof;   (iv) the industrial process of any of (i) to (iii), wherein the polypeptide is an enzyme or a catalytic antibody, or the polypeptide is immobilized;   (v) the industrial process of (i) to (iv), wherein the polypeptide is immobilized on an inorganic support or organic support; or the support comprises alumina, celite, Dowex-1-chloride, glass beads, silica gel, alginate hydrogel or alginate bead or equivalent; or the polypeptide is immobilized on an inorganic support or organic support; or the support comprises alumina, celite, Dowex-1-chloride, glass beads, silica gel, alginate hydrogel or alginate bead or equivalent; or the inorganic support or organic support comprises alumina, celite, Dowex-1-chloride, glass beads, silica gel, alginate hydrogel or alginate bead or equivalent;   (vi) the industrial process of (i) to (v), wherein at least one step is performed in a reaction vessel; or at least one step is performed in a reaction vessel comprising a gravitational gum separation device or a holding tank;   (vii) the industrial process of (i) to (vi), wherein at least one step is performed in a cell extract, or at least one step is performed in a whole cell;   (viii) the industrial process of (i) to (vii), wherein the chlorophyll-containing or chlorophyll-contaminated composition comprises a plant material, plant oil or plant extract; or the plant material, plant oil or plant extract comprises a vegetable oil or a seed oil; or the vegetable oil comprises a palm oil or a canola oil; or the plant material, plant oil or plant extract comprises an algae preparation;   (ix) the industrial process of (i) to (viii), wherein the method further comprises use of a lipoxygenase;   (x) the industrial process of (i) to (ix), wherein the chlorophyllase activity comprises catalyzing the cleavage of a tetrapyrrole macrocyclic ring and oxygenolytic cleavage of the macrocyclic ring;   (xi) the industrial process of (i) to (x), wherein the method further comprises removal of a chlorophilide generated by enzymatic degradation of a chlorophyll by adsorbing onto a silica gel or equivalent;   (xii) the industrial process of (i) to (xi), wherein the chlorophyll-containing or chlorophyll-contaminated composition comprises a textile or fabric; or the chlorophyll-containing or chlorophyll-contaminated composition comprises a wood or paper product or by-product; or the wood or paper product or by-product comprises a wood pulp or paper pulp; or the chlorophyll-containing or chlorophyll-contaminated composition comprises a non-wood paper product or by-product; or the non-wood or paper product comprises a rice paper;   (xiii) the industrial process of (i) to (xii), wherein the method further comprises a caustic neutralization step;   (xiv) the industrial process of (i) to (xiii), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a chlorophilide generated by the enzymatic degradation of the chlorophyll;   (xv) the industrial process of (i) to (xvi), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a pheophorbide generated by the enzymatic degradation of the pheophytin; or   (xvi) the industrial process of (i) to (xv), wherein the method further comprises addition of a phospholipase, or method further comprises addition of a phospholipase C;   (C) a degumming process comprising a step for enzymatic treatment of chlorophyll-containing or chlorophyll-contaminated compositions comprising:   (i) (a) providing a chlorophyll-containing or chlorophyll-contaminated composition;   (b) providing at least one polypeptide having a chlorophyllase activity or a chlorophyll catabolic enzyme activity; and   (c) reacting the composition of step (a) with the polypeptide of step (b) under conditions wherein the polypeptide can catalyze a chlorophyll-modifying reaction;   (ii) the degumming process of (i), wherein the polypeptide has the polypeptide sequence of  claim 54 ;   (iii) the degumming process of (i) or (ii), wherein the polypeptide comprises or is an enzyme or a catalytic antibody;   (iv) the degumming process of any of (i) to (iii), wherein the polypeptide is immobilized on an inorganic support or organic support; or the support comprises alumina, celite, Dowex-1-chloride, glass beads, silica gel, alginate hydrogel or alginate bead or equivalent; or the polypeptide is immobilized on an inorganic support or organic support; or the support comprises alumina, celite, Dowex-1-chloride, glass beads, silica gel, alginate hydrogel or alginate bead or equivalent; or the inorganic support or organic support comprises alumina, celite, Dowex-1-chloride, glass beads, silica gel, alginate hydrogel or alginate bead or equivalent;   (v) the degumming process of any of (i) to (iv), wherein at least one step is performed in a reaction vessel; or at least one step is performed in a reaction vessel comprising a gravitational gum separation device or a holding tank;   (vi) the degumming process of any of (i) to (v), wherein at least one step is performed in a cell extract; or at least one step is performed in a whole cell;   (vii) the degumming process of any of (i) to (vi), wherein the chlorophyll-containing or chlorophyll-contaminated composition comprises a plant material, plant oil or plant extract; or the plant material, plant oil or plant extract comprises a vegetable oil or a seed oil; or the vegetable oil comprises a palm oil or a canola oil; or the plant material, plant oil or plant extract comprises an algae preparation;   (viii) the degumming process of any of (i) to (vii), wherein the method further comprises use of a lipoxygenase;   (ix) the degumming process of any of (i) to (viii), wherein the chlorophyllase activity comprises catalyzing the cleavage of a tetrapyrrole macrocyclic ring and oxygenolytic cleavage of the macrocyclic ring;   (x) the degumming process of any of (i) to (ix), wherein the method further comprises removal of a chlorophilide generated by enzymatic degradation of a chlorophyll by adsorbing onto a silica gel or equivalent;   (xi) the degumming process of any of (i) to (x), wherein the chlorophyll-containing or chlorophyll-contaminated composition comprises a textile or fabric; or, the chlorophyll-containing or chlorophyll-contaminated composition comprises a wood or paper product or by-product; or, the wood or paper product or by-product comprises a wood pulp or paper pulp; or,   the chlorophyll-containing or chlorophyll-contaminated composition comprises a non-wood paper product or by-product; or, the non-wood or paper product comprises a rice paper;   (xii) the degumming process of any of (i) to (xi), wherein the method further comprises a caustic neutralization step;   (xiii) the degumming process of any of (i) to (xii), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a chlorophilide generated by the enzymatic degradation of the chlorophyll;   (xiv) the degumming process of any of (i) to (xiii), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a pheophorbide generated by the enzymatic degradation of the pheophytin;   (xv) the degumming process of any of (i) to (xiv), the method further comprises addition of a phospholipase, or the method further comprises addition of a phospholipase C; or   (xvi) the degumming process of any of (i) to (xv), the vegetable oil is a crude oil or a refined oil; or the vegetable oil is an undiluted crude oil preparation;   (D) enzymatically treating a chlorophyll-containing or chlorophyll-contaminated fabric comprising:   (i) (a) providing a detergent composition comprising at least one polypeptide having a chlorophyllase activity or a chlorophyll catabolic enzyme activity, wherein the activity comprises catalysis of a chlorophyll-modifying reaction, and;   (b) contacting the detergent composition with the chlorophyll-containing or chlorophyll-contaminated fabric under conditions wherein the polypeptide can catalyze a chlorophyll-modifying reaction;   (ii) the method of (i), wherein the polypeptide has the polypeptide sequence of  claim 54 , or is encoded by the nucleic acid of  claim 1 ;   (iii) the method of (i) or (ii), wherein the chlorophyll-modifying reaction comprises generation of chlorophyllide and phytol;   (iv) the method of any of (i) to (iii), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a chlorophilide generated by the enzymatic degradation of the chlorophyll;   (v) the method of any of (i) to (iv), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a pheophorbide generated by the enzymatic degradation of the pheophytin; or   (vi) the method of any of (i) to (v), wherein the method further comprises addition of a phospholipase, or the method further comprises addition of a phospholipase C;   (E) enzymatic treatment of pheophytin-containing or pheophytin-contaminated compositions comprising:   (i) (a) providing a pheophytin-containing or pheophytin-contaminated composition;   (b) providing at least one polypeptide having a chlorophyllase activity or a chlorophyll catabolic enzyme activity; and   (c) reacting the composition of step (a) with the polypeptide of step (b) under conditions wherein the polypeptide can catalyze a pheophytin-modifying reaction;   (ii) the method of (i), wherein the polypeptide has the polypeptide sequence of  claim 54 , or is encoded by the nucleic acid of  claim 1 ;   (iii) the method of (i) or (ii), wherein the chlorophyll-modifying reaction comprises generation of chlorophyllide and phytol;   (iv) the method of any of (i) to (iii), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a chlorophilide generated by the enzymatic degradation of the chlorophyll;   (v) the method of any of (i) to (iv), the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a pheophorbide generated by the enzymatic degradation of the pheophytin; or   (vi) the method of any of (i) to (v), the method further comprises addition of a phospholipase, or the method further comprises addition of a phospholipase C;   (F) enzymatic treatment of pheophytin-containing or pheophytin-contaminated compositions comprising:   (i) (a) providing a pheophytin-containing or pheophytin-contaminated composition;   (b) providing at least one polypeptide having a chlorophyllase activity or a chlorophyll catabolic enzyme activity; and   (c) reacting the composition of step (a) with the polypeptide of step (b) under conditions wherein the polypeptide can catalyze a pheophytin-modifying reaction;   (ii) the method of (i), wherein the polypeptide has the polypeptide sequence of  claim 54 , or is encoded by the nucleic acid of  claim 1 ;   (iii) the method of (i) or (ii), wherein the method further comprises the hydrolysis of a methyl ester on the chlorophyll by an esterase;   (iv) the method of any of (i) to (iii), wherein the method further comprises hydrolysis of a methyl ester on the pheophytin by an esterase;   (v) the method of any of (i) to (iv), wherein the method further comprises removal of the modified chlorophyll in an aqueous extraction;   (vi) the method of any of (i) to (v), wherein the method further comprises modifying pH to promote aqueous separation of chlorophyllide;   (vii) the method of any of (i) to (vi), wherein the method further comprises removal of the modified pheophytin in an aqueous extraction;   (viii) the method of any of (i) to (vii), wherein the method further comprises modifying pH to promote aqueous separation of pheophytin;   (ix) the method of any of (i) to (viii), wherein the method further comprises a caustic neutralization step;   (x) the method of any of (i) to (ix), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a chlorophilide generated by the enzymatic degradation of the chlorophyll;   (xi) the method of any of (i) to (x), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a pheophorbide generated by the enzymatic degradation of the pheophytin; or   (xii) the method of any of (i) to (xi), wherein the method further comprises addition of a phospholipase, or the method further comprises addition of a phospholipase C; or   (G) enzymatic treatment of chlorophyll-containing or chlorophyll-contaminated compositions comprising:   (i) (a) providing a chlorophyll-containing or chlorophyll-contaminated composition;   (b) providing a silica-immobilized chlorophyllase or chlorophyll catabolic enzyme; and   (c) reacting the composition of step (a) with the polypeptide of step (b) under conditions wherein the polypeptide can catalyze a chlorophyll-modifying reaction and the silica can adsorb the modified chlorophyll;   (ii) the method of (i), wherein the enzyme has the polypeptide sequence of  claim 54 , or is encoded by the nucleic acid of  claim 1 ;   (iii) the method of (i) or (ii), wherein the silica comprises a silica gel or equivalent, the silica comprises a TriSyl Silica or a SORBSIL R™ silica; or   (iv) the method of any of (i) to (iii), wherein the chlorophyllase comprises a silica-immobilized chlorophyllase.   
     
     
         170 - 202 . (canceled) 
     
     
         203 . A product of manufacture comprising
 (A) a degumming system for the enzymatic treatment of chlorophyll-containing or chlorophyll-contaminated compositions comprising:   (i) (a) a vegetable oil refining apparatus; and   (b) providing at least one polypeptide having a chlorophyllase activity or a chlorophyll catabolic enzyme activity, wherein the activity comprises catalysis of a chlorophyll-modifying reaction, and the vegetable oil refining apparatus can react a chlorophyll-containing or chlorophyll-contaminated composition with the polypeptide to under conditions wherein the polypeptide can catalyze a chlorophyll-modifying reaction;   (ii) the product of manufacture of (i), wherein the polypeptide has the polypeptide sequence of  claim 54 , or is encoded by the nucleic acid of  claim 1 ;   (iii) the product of manufacture of (i) or (ii), wherein the vegetable oil refining apparatus comprises an oil leaving expellor, a holding tank or a gravitational gum separation device;   (iv) the product of manufacture of any of (i) to (iii), wherein the chlorophyll-modifying reaction comprises generation of chlorophyllide and phytol;   (v) the product of manufacture of any of (i) to (iv), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a chlorophilide generated by the enzymatic degradation of the chlorophyll;   (vi) the product of manufacture of any of (i) to (v), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a pheophorbide generated by the enzymatic degradation of the pheophytin;   (vii) the product of manufacture of any of (i) to (vi), wherein the method further comprises addition of a phospholipase, or the method further comprises addition of a phospholipase C; or   (viii) the product of manufacture of any of (i) to (vii), wherein the chlorophyllase comprises a silica-immobilized chlorophyllase, and optionally the silica comprises a silica gel or equivalent, and optionally the silica comprises a TriSyl Silica or a SORBSIL R™ silica; or   (B) a detergent for an enzymatic treatment of a chlorophyll-containing or a chlorophyll-contaminated fabric comprising:   (i) (a) a detergent composition; and   (b) at least one polypeptide having a chlorophyllase activity or a chlorophyll catabolic enzyme activity, wherein the activity comprises catalysis of a chlorophyll-modifying reaction;   (ii) the detergent composition of (i), wherein the polypeptide has a sequence as set forth in  claim 54 , or is encoded by a nucleic acid as set forth in  claim 1 ;   (iii) the detergent composition of (i) or (ii), wherein the chlorophyll-modifying reaction comprises generation of chlorophyllide and phytol;   (iv) the detergent composition of any of (i) to (iii), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a chlorophilide generated by the enzymatic degradation of the chlorophyll;   (v) the detergent composition of any of (i) to (iv), wherein the method further comprises an adsorbent-free or reduced adsorbent silica refining step to remove a pheophorbide generated by the enzymatic degradation of the pheophytin; or   (vi) the detergent composition of any of (i) to (v), wherein the method further comprises addition of a phospholipase, or the method further comprises addition of a phospholipase C.   
     
     
         204 - 237 . (canceled)