Enzymatic removal of chlorophyll substrates from triacylglycerol-based oils

Abstract

The present invention relates to a process for treating an oil comprising a chlorophyll substrate, the process comprising contacting the oil with a polypeptide having decolorase activity or a composition comprising the polypeptide, wherein the polypeptide is selected from the group consisting of: a. a polypeptide which has at least 80% identity to amino acids 1 to 318 of SEQ ID NO: 1; and, b. a polypeptide encoded by a nucleic acid sequence that has at least 80% identity to the nucleic acid sequence of SEQ ID NO: 2.

Claims

1. A process for treating an oil comprising a chlorophyll substrate, the process comprising contacting the oil with a polypeptide having decolorase activity or a composition comprising the polypeptide, wherein the polypeptide is dosed into the oil in an amount of 1 to 50 U/gram oil, wherein the treatment reduces the total concentration of chlorophyll substrates in the oil by at least 5% by weight, compared to the total concentration of chlorophyll substrates in the oil prior to treatment.

2. The process of claim 1, wherein the polypeptide has chlorophyllase activity, pheophytinase activity, pyropheophytinase activity, or combinations thereof.

3. The process of claim 1, wherein the oil comprises a triacylglycerol-based oil selected from the group consisting of canola oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil, hempseed oil, linseed oil, mango kernel oil, meadowfoam oil, neatsfoot oil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil, rapeseed oil, rice bran oil, safflower oil, sasanqua oil, sesame oil, soybean oil, sunflower seed oil, tall oil, tsubaki oil, vegetable oil, and an oil from algae.

4. The process of claim 1, wherein the oil comprises an oil from algae.

5. The process of claim 1, wherein the oil comprises an oil selected from the group consisting of a crude non-degummed oil, a degummed oil, a caustic refined oil, a caustic refined and water washed oil, or a water degummed oil.

6. The process of claim 1, wherein the treatment reduces the total concentration of chlorophyll substrates in the oil by at least 50% by weight, compared to the total concentration of chlorophyll substrates in the oil prior to treatment.

7. The process of claim 1, wherein the chlorophyll substrate comprises pyropheophytin, and at least a portion of the pyropheophytin is converted into pyropheophorbide.

8. The process of claim 7, wherein the treatment reduces the concentration of pyropheophytin in the oil by at least 5% by weight, compared to the total concentration of pyropheophytin in the oil prior to treatment.

9. The process of claim 8, wherein the treatment reduces the concentration of pyropheophytin in the oil by at least 50% by weight, compared to the total concentration of pyropheophytin in the oil prior to treatment.

10. The process of claim 1, wherein the chlorophyll substrate comprises pheophytin, and at least a portion of the pheophytin is converted into pheophorbide.

11. The process of claim 10, wherein the treatment reduces the concentration of pheophytin in the oil by at least 5% by weight, compared to the total concentration of pheophytin in the oil prior to treatment.

12. The process of claim 11, wherein the treatment reduces the concentration of pheophytin in the oil by at least 50% by weight, compared to the total concentration of pheophytin in the oil prior to treatment.

13. The process of claim 1, wherein the chlorophyll substrate comprises chlorophyll, and at least a portion of the chlorophyll is converted into chlorophyllide.

14. The process of claim 13, wherein the treatment reduces the concentration of chlorophyll in the oil by at least 5% by weight, compared to the total concentration of chlorophyll in the oil prior to treatment.

15. The process of claim 14, wherein the treatment reduces the concentration of chlorophyll in the oil by at least 50% by weight, compared to the total concentration of chlorophyll in the oil prior to treatment.

16. The process of claim 1, wherein the polypeptide is contacted with the oil at a pH of from 2 to 10.

17. The process of claim 16, wherein the polypeptide is contacted with the oil at a pH of from 4.0 to 5.0.

18. The process of claim 1, wherein the polypeptide is contacted with the oil for from 1.5 hours to 6 hours.

19. The process of claim 18, wherein the polypeptide is contacted with the oil for 2 hours.

20. The process of claim 1, comprising contacting the oil with the polypeptide and water, and stirring for from 0.5 to 24 hours, wherein the oil comprises a non-degummed crude oil.

21. The process of claim 1, comprising contacting the oil with the polypeptide, water, and an additional enzyme, and stirring for from 0.5 to 24 hours, wherein the oil comprises a non-degummed crude oil.

22. The process of claim 21, wherein the additional enzyme is selected from the group consisting of PLC, PI-PLC, and combinations thereof.

23. The process of claim 1, comprising contacting the oil with the polypeptide and water, and stirring the resulting oil, wherein the oil comprises a once refined oil.

24. The process of claim 1, further comprising treating the oil with an additional enzyme selected from the group consisting of a phospholipase, a pheophytinase, a pyropheophytinase, a chlorophyllase, and combinations thereof.

25. A process for treating an oil comprising a chlorophyll substrate, the process comprising contacting the oil with a polypeptide having decolorase activity or a composition comprising the polypeptide, water, and an additional enzyme, at a pH of from 4 to 8; and stirring the resulting oil for from 0.5 to 24 hours, wherein the polypeptide is dosed into the oil in an amount of 1 to 50 U/gram oil.

26. The process of claim 25, wherein the additional enzyme is selected from the group consisting of PLC, PI-PLC and combinations thereof.

27. The process of claim 25, further comprising adding a PLA enzyme to the oil following stirring.

28. The process of claim 27, wherein the PLA enzyme is a PLA1 enzyme.

29. The process of claim 28, wherein the oil is stirred for from 1 to 8 hours following addition of the PLA1 enzyme.

Description

FIGURES

(1) FIG. 1: Overview of the conversion of chlorophyll into pheophytin and pyropheophytin and into the respective reaction products chlorophyllide, pheophorbide and pyropheophorbide. The A compounds are shown, which have a methyl group at the C7 position. B compounds have an aldehyde in the C7 group instead of a methyl group. Structures are taken from PubChem, NCBI.

(2) FIG. 2: HPLC results of incubation pheophytin a and b and pyropheophytin a and b with different putative chlorophyllases at pH 7 and 50? C., for 24 hours. The amounts of the substrates pheophytin a and b and pyropheophytin a and b and the reaction products pheophorbide a and b and pyropheophorbide a and b are given as peak surface areas. The first two columns show the sum of reaction products and substrates. nd means: not detectable.

(3) FIG. 3: HPLC results of incubation pheophytin a and b and pyropheophytin a and b with different putative chlorophyllases at pH 5 and 50? C., for 24 hours. The amounts of the substrates pheophytin a and b and pyropheophytin a and b and the reaction products pheophorbide a and b and pyropheophorbide a and b are given as peak surface areas. The first two columns show the sum of reaction products and substrates. nd means: not detectable.

(4) FIG. 4: a) Chlorophyll derivatives b) Phosphor compounds in canola oil after 24 h incubation with CHL26 enzyme from Hordeum vulgare or the reference enzyme ELDC94 from Chlamydomonas reinhardtii.

(5) FIG. 5: a) Chlorophyll derivatives, and b) Phosphor compounds in canola oil and soy bean after several incubations with CHL26 enzyme from Hordeum vulgare and/or the reference enzyme ELDC94 from Chlamydomonas reinhardtii, under different reaction conditions and c) chlorophyll derivatives in the obtained gums.

(6) FIG. 6: Chlorophyll derivatives in canola oil and soybean oil after caustic refining and after incubation with CHL26 enzyme from Hordeum vulgare or the reference enzyme ELDC94 from Chlamydomonas reinhardtii

(7) FIG. 7: Schematic presentation of a chemical refinery process for triacylglycerol based oils. A process of solvent extraction and/or pressing on an oilseed (rapeseed or soybean), oil fruit plant (palm), or single cell source (algal) to obtain a crude oil. The crude oil this then treated with citric or phosphoric acid to react with the non-hydratable phospholipids and then the addition of sodium hydroxide to neutralize the free fatty acids and form sodium soaps. The sodium soaps form an emulsion with the water present allowing the removal of non-hydratable phospholipids when the oil is centrifuged to produce refined oil. The refined oil may then be washed with hot water and centrifuged to remove the remaining soaps and phospholipids. Alternatively, the refined oil may be treated with acidic silica to adsorb soaps, trace metals and phospholipids. The industrial acidic silicas do not have any capacity to remove chlorophyll or chlorophyll derivatives. The oil is then treated with bleaching earth to remove the soaps, phospholipids, and chlorophyll and chlorophyll derivatives present in the oil. The final step in the deodorization step of steam distillation at elevated temperatures and vacuums of less than 5 mBar. The distillation primarily removes peroxides, aldehydes, ketones and other flavor compounds. It also destroys beta-carotene and removes the remaining free fatty acids (0.1 percent) to reach a level of 0.02 to 0.05% final Free Fatty Acid (FFA).

(8) FIG. 8: Schematic presentation of an enzymatic degumming/physical refining process. The crude oil is treated with phosphoric or citric acid to enable the non-hydratable phospholipids to lose the calcium or magnesium bond to them at a pH of roughly 2. The sodium hydroxide is then added to bring the pH above 4 for citric acid or above 6 for phosphoric acid in order that the phospholipase may work and obtain a very low residual phosphorus <5 ppm) after the enzymatic reaction with the PLAs. Alternatively, the PLAs may be reacted with the PLC and/or PI-PLC to maximize the oil yield and still obtain a very low residual phosphorus allowing for physical refining. The oil is then either washed or treated with an acidic silica followed or in combination with bleaching earth. After the bleaching process with chlorophyll levels of less than 50 ppb, the oil is physically refined in the deodorizer. The high temperature steam distillation removes all of the compounds describe above in FIG. 7, but its primary purpose is the removal of FFA. The FFAs are distilled and collected in the scrubber. Very limited neutral oil is lost in the deodorization process compared to the losses associated from the emulsions formed in water degumming or chemical refining.

(9) FIG. 9: Schematic presentation of the use of a decolorase enzyme in the water degumming process or the enzyme assisted water degumming process. A decolorase enzyme may be added with the water at 60? C., or with the PLC, or with the combination of PLC and PI-PLC. After two hours of incubation, the oil is heated to 70 to 85? C. and centrifuged to remove the reacted gums and reacted chlorophyll derivatives.

(10) FIG. 10: Schematic presentation of an enzymatic degumming process with a decolorase enzyme. The crude oil is first treated with citric acid to a pH of roughly 2 to dissociate the bond calcium and magnesium ions, the pH is raised above 4 to enable the PLCs and Decolorase enzymes in a pH that enable them to work efficiently. 1 to 5 percent water is added for the hydrolysis reactions. After the completion of the PLCs and Decolorase incubations, a PLA1 or PLA2 may be added to react with the non-hydratable phospholipids present in the oil. After an additional incubation of 2 to 6 hours, the oil is heated to 70 to 85? C. and centrifuged to remove the reacted gums and chlorophyll derivatives producing an oil with less than 5 ppm residual phosphorus in the oil.

(11) FIG. 11: Schematic presentation of a chemical refining process with a decolorase enzyme. The decolorase enzyme may not be added in the acid or caustic addition steps due to the very low pH (roughly 2) and the very high pH (roughly 14) in the early steps of the process. The decolorase enzyme must be added after the initial centrifuge step in the refined oil. It is advantageous to add the decolorase enzyme with the washing step at a temperature suitable for the enzyme (50 to 65? C.). Allow an incubation time of at least two hours followed by heating to 70 to 85? C. prior to centrifugation. The oil would then be further processed.

SEQUENCES

(12) SEQ ID NO: 1=CHL26 polypeptide having decolorase including a pyropheophytinase activity from Hordeum vulgare.

(13) TABLE-US-00001 MASAGDVFDHGRHGTSLARVEQAKNTRCSAASRVDADAQAQQSPPKPLLV AAPCDAGEYPVVVFLHGYLCNNYFYSQLIQHVASHGFIVVCPQLYTVSGP DTTSEINSAAAVIDWLAAGLSSKLAPGIRPNLAAVSISGHSRGGKVAFAL GLGHAKTSLPLAALIAVDPVDGTGMGNQTPPPILAYKPNAIRVPAPVMVI GTGLGELPRNALFPPCAPLGVSHAAFYDECAAPACHLVARDYGHTDMMDD VTTGAKGLATRALCKSGGARAPMRRFVAGAMVAFLNKWVEGKPEWLDAVR EQTVAAPVVLSAVEFRDE SEQ ID NO: 2: Codon optimized nucleic acid sequence encoding a polypeptide having decolorase including pyropheophytinase activity from Hordeum vulgare CHL26 for expression in Pseudomonas fluorescens. SEQ ID NO: 3; CHL25 putative chlorophyllase from Gossypium raimondii SEQ ID NO: 4; CHL27 putative chlorophyllase from Phoenix dactylifera SEQ ID NO: 5; CHL28 putative chlorophyllase from Wollemia nobilis SEQ ID NO: 6; CHL29 putative chlorophyllase from Cucumis sativus SEQ ID NO: 7; CHL30 putative chlorophyllase from Tarenaya hassleriana SEQ ID NO: 8; CHL31 putative chlorophyllase from Solanum tuberosum SEQ ID NO: 9; CHL32 putative chlorophyllase from Populus trichocarpa SEQ ID NO: 10; CHL33 putative chlorophyllase from Vigna radiata SEQ ID NO: 11; N1 Negative control, Green Fluorescent Protein (GFP) SEQ ID NO: 12; P2, Chlamydomonas reinhardtii chlorophyllase having pyropheophytinase activity. SEQ ID NO: 12 is also referred to herein as ELDC94. SEQ ID NO: 13 SpeI site and ribosome binding site SEQ ID NO: 14 stop codon and XhoI site

EXAMPLES

Materials and Methods

(14) General

(15) Standard genetic techniques, such as overexpression of enzymes in the host cells, genetic modification of host cells, or hybridisation techniques, are known methods in the art, such as described in Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual (3.sup.rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York (1987). Water is Milli-Q water where nothing else is specified.

(16) Analytical Methods:

(17) pHstoichiometric addition of acid and base to a water percentage that was added to the oil. 2 percent water in a 2000 grams reaction would be 40 grams, adding 2.0 grams of a 50 percent solution of citric acid, plus 1.6 mL of 4 M sodium hydroxide would yield a water solution with a pH of 4.5. The pH of the oil will always remain 7.

(18) SoapAmerican Oil Chemists' Society Official Method Cc 13a-43, revised 2017. Free Fatty AcidAmerican Oil Chemists' Society Official Method Ca 5a-40, revised 2017.

(19) ColorAmerican Oil Chemists' Society Official Method Ce 13e-92, reapproved 2017. Utilized Tintometer's PFX-950 at 5V cell.

(20) Phosphorus and trace metalsAmerican Oil Chemists' Society Official Method Ca 17-01-43, revised 2017. Phospholipid Compositions For .sup.31P NMR methods (also referred to as 31-P NMR), 10 ?L of 10% DOL dispersion was dispersed in 1 mL of an aqueous solvent containing demineralized water with 10% deuterium oxide (D20, Cambridge Isotope Laboratories, DLM-4), 25 mg/mL deoxycholic acid (Sigma D2510), 5.84 mg/mL EDTA di Na (Titriplex III, Merck 108418), and 5.45 mg/mL TRIS base (Tris(hydroxymethyl) aminomethane, Merck 108387), of which the pH was adjusted to pH 9 using 4N KOH and to which 2 mg/mL TIP internal standard (tri-isopropylphosphate, Aldrich 554669) (accurately weighed) was added.

(21) All samples were measured in a Bruker 400 MHz AvanceIII NMR spectrometer with a Prodigy BBO probe. The temperature of the probe head was set at 300K.

(22) The measurement for quantification was performed with semi-quantitative parameters: 128 scans, 90? pulse, D1=5 sec. Values are reported in ?mol/g of dry weight (DOL) of the sample.

(23) Analysis of Coloured Compounds by HPLC-FLU

(24) The analysis of pheophytins A and B, and pyropheophytins A and B, and their phorbides was performed by HPLC using fluorescence detection, a method developed based on the work of Hwang et al J. Food Hyg. Soc. Japan Vol. 46, No. 2, 45-48, extended by fluorescence detection at ?ex 410 nm/?em 666 nm for the A compounds, and ?ex 436 nm/?em 653 nm for the B compounds.

(25) Sample Preparation

(26) Oil samples were diluted in acetone, 1 g oil in 9 mL acetone, and centrifuged at 14000 rpm for 5 minutes. The clear supernatants were transferred into injection vials, and 10 ?l of a sample was injected into the HPLC. As the chlorophyll levels were so low in all practical oil samples, these were not taken into account in the analysis.

(27) Data Analysis

(28) The peak surface areas (in arbitrary units) of the chromatograms indicate the amount of pheophytins, pyropheophytins, pheophorbides and pyropheophorbides present in the oil samples. FIGS. 2 and 3 show the peak surface areas of pheophytins, pyropheophytins, pheophorbides and pyropheophorbides in oil samples after incubation with putative chlorophyllases at pH 5 and pH 7. The sum of the peak surface area of phytines, the sum of peak surface area of phorbides and the peak surface area of the individual compounds are shown. The formation of pheophorbide and pyropheophorbide is a measure for the presence of pheophytinase activity and pyropheophytinase activity, respectively.

(29) Enzymes

(30) Purifine? Phospholipase C (PLC), and Purifine? PI-PLC and a fungal PLA1 were obtained from DSM.

(31) Purifine? Phospholipase C comprises amino acids 38-282 of SEQ ID NO: 2, having the amino acid substitutions 63D, 131S and 134D disclosed in WO2005/086900

(32) Purifine? PI-PLC comprises the mature polypeptide according to SEQ ID NO: 8 disclosed in WO2011/046812.

(33) Fungal PLA1 comprises the mature amino acid sequence of SEQ ID NO: 1 disclosed in European application no. EP18171015.3

(34) Equipment

(35) The overhead mixer was an IKA RW 20 Digital with a flat blade paddle. The centrifuge was a De Laval GyroTester installed with The Bowl Unit for continuous separation. The centrifuge bowl was closed with the plug screws installed.

(36) Shear mixing was accomplished with an Ultra-Turrax homogenizer SD-45 with a G450 rotor stator at 10,000 rpm.

Example 1. Expression of a Putative Chlorophyllases in Pseudomonas

(37) Putative chlorophyllases (CHL) as provided in the tables of FIGS. 2 and 3 were expressed in the Pseudomonas system obtained from Dow Global Technologies Inc. (US20050130160, US20050186666 and US20060110747). The 12 synthetic genes based on the protein sequence of the putative chlorophyllases protein sequences as shown in FIGS. 2 and 3 were designed by optimizing the gene codon usage for Pseudomonas according to the algorithm of DNA2.0 (GeneGPS? technology). For cloning purposes, the DNA sequence contain a SpeI site and ribosome binding site (ACTAGTAGGAGGTAACTAATG) (SEQ ID NO: 13) at the 5-end and a stop codon and XhoI site (TGATGACTCGAG) (SEQ ID NO: 14) at the 3-end.

(38) SEQ ID NO: 2 shows the codon optimized nucleic acid sequence encoding the putative chlorophyllase SEQ ID NO:1 of Hordeum vulgare.

(39) The DNA sequences were inserted in the pDOW1169 vector (Dow Global Technologies Inc., US20080058262) using SpeI and XhoI restriction enzyme cloning. The pDOW1169 vectors containing the genes encoding the CHL and PPH enzymes under control of a modified lac promotor were then introduced into Pseudomonas fluoresce/1s uracil auxotrophic strain DPfl0. The transformed cells were selected after incubating on M9 minimal medium at 30? C. for 48 hours (Dow Global Technologies Inc., US20050186666) without uracil (Schneider et al. 2005).

(40) Correct transformants were pre-cultured in 24 well pre-sterile deep well plates (Axygen, CA, USA) containing 3 ml M9 medium. Plates were covered by a Breathseal (Greiner bio-one, Frickenhausen, Germany) and incubated at 30? C., 550 rpm and 80% humidity for 16 hours in a Microton incubator shaker (Infors AG, Bottmingen, Switzerland). From these cultures 30 ?I was used to inoculate a second 24 well pre-sterile deep well plates (Axygen, CA, USA) containing 3 ml M9 medium at 30? C., 550 rpm for 24 hours. After 8 hours, the cultures were induced with IPTG (0.3 mM final concentration). Cultures were harvested by centrifugation for 10 minutes at 2750 rpm and the supernatants removed. The cell pellets were stored overnight at ?20? C. The cell pellets from the 3 ml cultures were suspended in 1 ml lysis buffer and incubated for one hour at 37? C. Lysis buffer (1 mM EDTA, 50 mM Tris, pH 8, 0.25 mg/ml lysozyme, 10 mg/ml DnaseI, 25 ?M MgSO.sub.4 and 0.03% triton). The lysates were centrifuged at 2750 rpm for 10 minutes and the supernatants were removed and stored.

Example 2. Determination of Pyropheophytinase Activity in Cell-Free Extracts in Crude Canola Oil

(41) Incubation

(42) Crude canola oil from North American origin, high in pheophytins and pyropheophytins was used to determine activity of the enzyme in the supernatant as produced in Example 1 on pyropheophytin A and B in the following way. Buffer (5% (v/v)) was added to oil under high-shear mixing using a Silverson mixer. For pH 5, a 20 mM citric acid buffer was used. For pH 7 a 20 mM phosphate buffer was used. A 24 wells microtiter plate was filled with 1.425 mL buffer-in-oil dispersion per well, and to each well 75 ?L, 5% (v/v) cell-free extract (supernatant) produced in Example 1 was added. A list of tested samples is given in the tables of FIG. 2 and FIG. 3, and include a positive reference containing Chlamydomonas reinhardtii pyropheophytinase and negative control Green Fluorescent Protein (GFP). The microtiter plate was covered with plastic foil [Fasson S695]. Each well was stirred with an individual magnetic stirring bar. Incubations were performed at 50? C. using a KBMD microtiter-plate stirrer. Samples were taken after 24 hours and analysed for the presence of pheophytins A and B, and pyropheophytins A and B, and their phorbides using HPLC-FLU as described above.

(43) The results in FIGS. 2 and 3 show that only CHL26, a putative chlorophyllase from Hordeum vulgare, was able to hydrolyse all pheophytins and pyropheophytins into their respective (pyro)pheophorbides at pH 7 and pH 5.

Example 3. Incubation of Crude Canola Oil with CHL26 Versus Time

(44) Incubation of crude canola oil with 5% cell free extract of Hordeum vulgare putative chlorophyllase CHL26 produced as described in Example 1, was repeated in the same way as described in Example 2 at pH7. Samples were taken after 30 min, 2 hr, 5 hr, and 24 hr. Pyropheophytin a and b, and pheophytin a and b, pyropheophorbide a and b and pheophorbide a and b were measured by HPLC as described above.

(45) The formation of the reaction products pyropheophorbide a and b and pheophorbide a and b in Table 1 is expressed as percentage of the amount reaction product (respective phorbide molecule) after 24 hr.

(46) Table 2 shows the relative amounts of pheophytins and pyropheophytins as a function of time after 0.5, 2 and 5 hr, expressed in percentages relatively to the value at t=0 (average of 4 measurements).

(47) TABLE-US-00002 TABLE 1 Relative HPLC results for all reaction products after incubation for 0.5, 2, 5 and 24 hours at pH 7 and 50? C., in percentages relative to value after 24 hrs. Time Pheophorbide Pyropheophorbide Pheophorbide Pyropheophorbide [hr] B (%) B (%) A (%) A (%) 0.5 53.7 48.8 69.4 56.7 2 85.3 95.5 96.4 90.0 5 92.9 92.9 98.7 94.3 24 100.0 100.0 100.0 100.0

(48) TABLE-US-00003 TABLE 2 Relative HPLC results for all phytin compounds after incubation for 0.5, 2, and 5 hours at pH 7 and 50? C., in percentages relative to value at t = 0. Time Pheophytin Pyropheophytin Pheophytin Pyropheophytin Sum [hr] B (%) B (%) A (%) A (%) phytins (%) 0 100.0 100.0 100.0 100.0 100.0 0.5 34.7 43.8 34.4 46.4 41.4 2 0.0 12.7 0.0 12.9 8.2 5 0.0 6.2 0.0 6.0 3.9

(49) The results in Table 1 and 2 show that enzyme CHL26 from Hordeum vulgare is able to hydrolyse both pheophytin and pyropheophytin, and both the a and b compounds. After 2 hrs all pheophytins were converted (below detection limit), whereas after 5 hours almost all the pyropheophytins were converted.

Example 4. Production of CHL26 and ELDC94 by 10 L Bioreactor Fermentation

(50) Strains and Inoculum

(51) Of a P. fluorescens strain containing CHL26 (SEQ ID NO: 1) and Chlamydomonas reinhardtii (ELDC94; SEQ ID NO: 12) chlorophyllase as described in Example 1 a pre-culture was prepared in one-phase shake flasks with complex medium comprising yeast extract, slats and glycerol as a C-source, which was used as inoculum for the 10 L fermentations with inoculation ratio of 10% described below.

(52) 10 L Fermentations

(53) Fermentation process was based on industrial Pseudomonas fluorescens fermentations (fed-batch process, sugar limited, IPTG induced). The fermentation process consisted of biomass production under exponential feed of glucose as C-source followed by production phase under IPTG induction system. After 23 hr fermentation (end of biomass production phase), IPTG was added to a final concentration of 0.125 mM in order to induce enzyme production. The feed rate of C-source (glucose) was reduced to ?70% of maximum and fermentation prolonged till 48-55 hours after inoculation.

(54) At the end of fermentation, the broth was killed off and the enzyme release via benzoate treatment followed by pH increase of the fermentation broth.

(55) Recovery

(56) The intra-cellular enzyme was released by homogenization. Two passes at 750 bars, with a cooling period of 12-hours in-between was applied. Subsequently the homogenized broth was diluted with 30% water, 15% DBF (Dicalite BF), Calcium Chloride (20 g/kg original broth), and Flocculent C577 (0.1% on original broth) were added. The pH was adjusted to 8, and the material was clarified and ultra-filtrated. The UF was stabilized with 50% glycerol, and to ensure full killing of remaining bacteria MEP (methyl/ethyl paraben in a solution with propene-diol) was added, diluting the product with about 15% v/v.

(57) Activity

(58) Activity on p-NP Substrates

(59) The enzyme activity was determined using the chromogenic substrate 4-nitrophenyl butyrate (Sigma N9874). Substrate stock solution: 50 mM pNP-butyrate in acetonitrile. Substrate solution: Prior to use the substrate stock solution was mixed in ratio 1:4 with 0.1 M phosphate buffer pH 7.0 also containing 0.2% BSA and 2.0% Triton X-100.

(60) In micro titer plates, 120 ?L phosphate buffer (same as above) was mixed with 15 ?L substrate solution and equilibrated at 37? C. After starting the reaction by adding 15 sample, the OD at 405 nm was measured for 5 minutes. Also, a blank measurement was done by adding 15 ?L buffer instead of sample. The slope of the linear part of the curve is used as measure for the activity. Samples were diluted such to assure that the absorbance increase after 5 minutes is less than 1.0.

(61) Activity is calculated as follows:
U/mL=(?Abs/min sample??Abs/min blank)/(?.sub.pNP?5)?1000?150/15?Df/W ?.sub.pNp=Molar Extinction Coefficient of para-nitro-phenol [L.Math.mol-1.Math.cm-1] 5=Incubation time [min] 1000=factor from mmol to ?mol 150=assay volume [?L] 15=sample volume [?L] Df=Dilution factor W=weight of sample (g)

(62) The activity is expressed as the amount of enzyme that liberates 1 micromol p-nitrophenol per minute under the conditions of the test. Calibration is done using a 4-nitrophenol standard solution (Sigma N7660) diluted in the above-mentioned phosphate buffer.

(63) The activity of the final formulations of CHL26 was 1.4 U/g (0.5 w/w %), and of ELDC94 87 U/g (0.04 w/w %).

Example 5. Incubation of Crude Canola Oil with an Enzyme Having Pyropheophytinase Activity Derived from Hordeum vulgare (CHL26) Compared to Incubation of Crude Canola Oil with a Reference Enzyme (ELDC94) from Chlamydomonas reinhardtii at Various Conditions

(64) Crude canola oil was incubated with 0.5 w/w % cell free extract of Hordeum vulgare putative chlorophyllase CHL26 and compared to 0.04 w/w % of cell-free extract of Chlamydomonas reinhardtii chlorophyllase (coded ELDC94=Ref) both enzymes produced as described in Example 4. The incubation was performed on 10 g scale (10 g oil in 15 ml glass reaction vessels incubated on a hot plate aluminium reaction block with temperature control. Contents are kept vigorously stirred by magnetic bars), and now at three different temperatures (40, 50 and 60? C.), and under four regimes with varying acidity of the aqueous phase: Acidic: 400 ppm citric acid pre-treatment; Mildly acidic: pre-treatment with 500 ppm citric acid and 138 ppm caustic (NaOH); Neutral: only water; Mildly alkaline: pre-treatment with 150 ppm NaOH.

(65) The total water level during incubation is 3% w/w, which includes enzyme formulation and pre-treatment solutions. Prior to the experiment, the acidity of the aqueous environment was assessed by diluting the pre-treated oil 1:1 with water and then the pH was measured by a pH meter. This resulted in the following pH values, indicative for the acidity of the aqueous environment in the dispersion during reaction: Acidic: pH 3.4; mildly acidic: pH 4.5; Neutral: pH 5.9 and alkalic pH 7.9.

(66) For pre-treatment with citric acid, the citric acid (as 50% w/w solution) was added to the oil at 70? C., kept stirred at 70? C. for 30 minutes, subsequently the temperature was reduced to incubation temperature and for the mildly acid condition the NaOH (as 2.0% w/w solution) was added. In case of only NaOH addition, the oil was stirred at incubation temperature for 30 minutes.

(67) During incubation, samples were taken after 0.5, 2, 4 and 24 hours, and analysed by HPLC-Flu as described in Example 2, now against a set of standards with known concentration. Concentrations of all substrates (chlorophyll, pheophytins, pyropheophytina and b) and all reaction products (chlorophyllide, pheophorbide, pyropheophorbidea and b) were summed into total substrates and total reaction products, respectively, in mg/kg oil. All results are given in percentage of substrates and reaction products in the table below.

(68) The results in Tables 3, 4 and 5 show that the Hordeum vulgare enzyme CHL26 according to SEQ ID NO: 1 has a wider application range in the presence of acid and caustic and is active at a higher temperature than the reference chlorophyllase from Chlamydomonas reinhardtii.

(69) TABLE-US-00004 TABLE 3 Chlorophyll derivatives (wt %) in crude canola oil after incubation with the CHL26 enzyme from Hordeum vulgare or the reference enzyme ELDC94 from Chlamydomonas reinhardtii at different conditions at 40? C. CHL26 Reference 40? C. Time Sum Sum reaction Sum Sum reaction Condition [hr] substrates products substrates products 0 94.9 5.1 94.9 5.1 Acidic 0.5 66.5 33.5 80.6 19.4 2 56.4 43.6 78.1 21.9 4 51.9 48.1 75.2 24.8 24 45.4 54.6 75.2 24.8 Mildly 0.5 29.1 70.9 35.2 64.8 acidic 2 10.0 90.0 28.7 71.3 4 3.4 96.6 19.0 81.0 24 0.0 100.0 15.4 84.6 Neutral 0.5 30.9 69.1 3.2 96.8 2 10.0 90.0 1.7 98.3 4 4.4 95.6 1.8 98.2 24 3.0 97.0 1.7 98.3 Mildly 0.5 71.8 28.2 60.7 39.3 alkaline 2 72.5 27.5 55.2 44.8 4 57.4 42.6 38.2 61.8 24 14.8 85.2 32.8 67.2

(70) TABLE-US-00005 TABLE 4 Chlorophyll derivatives (wt %) in crude canola oil after incubation with the CHL26 enzyme from Hordeum vulgare or the reference enzyme ELDC94 from Chlamydomonas reinhardtii at different conditions at 50? C. CHL26 Reference 50? C. Time Sum Sum reaction Sum Sum reaction Condition [hr] substrates products substrates products 0 94.9 5.1 94.9 5.1 Acidic 0.5 87.7 12.3 89.9 10.1 2 87.7 12.3 92.6 7.4 4 88.5 11.5 93.4 6.6 24 88.0 12.0 92.6 7.4 Mildly 0.5 27.5 72.5 21.3 78.7 acidic 2 9.7 90.3 11.7 88.3 4 2.7 97.3 8.7 91.3 24 2.2 97.8 2.1 97.9 Neutral 0.5 28.5 71.5 5.8 94.2 2 13.7 86.3 3.9 96.1 4 5.5 94.5 2.0 98.0 24 0.6 99.4 0.6 99.4 Mildly 0.5 66.8 33.2 54.6 45.4 alkaline 2 66.4 33.6 62.1 37.9 4 51.1 48.9 51.8 48.2 24 10.9 89.1 41.0 59.0

(71) TABLE-US-00006 TABLE 5 Chlorophyll derivatives (wt %) in crude canola oil after incubation with the CHL26 enzyme from Hordeum vulgare or the reference enzyme ELDC94 from Chlamydomonas reinhardtii at different conditions at 60? C. CHL26 Reference 60? C. Time Sum Sum reaction Sum Sum reaction Condition [hr] substrates products substrates products 0 94.9 5.1 94.9 5.1 Acidic 0.5 90.5 9.5 94.7 5.3 2 90.5 9.5 95.0 5.0 4 90.7 9.3 92.7 7.3 24 90.5 9.5 94.9 5.1 Mildly 0.5 13.5 86.5 60.4 39.6 acidic 2 2.5 97.5 65.7 34.3 4 4.1 95.9 66.4 33.6 24 2.0 98.0 68.5 31.5 Neutral 0.5 29.0 71.0 11.9 88.1 2 10.1 89.9 7.4 92.6 4 5.2 94.8 4.6 95.4 24 0.0 100.0 1.5 98.5 Mildly 0.5 57.7 42.3 52.5 47.5 alkaline 2 65.1 34.9 80.4 19.6 4 67.4 32.6 80.4 19.6 24 33.0 67.0 93.0 7.0

Example 6. Incubation of Solvent Extracted Crude Canola Oil with an Enzyme Having Pyropheophytinase Activity Derived from Hordeum vulgare (CHL26) Compared to a Reference Enzyme from Chlamydomonas reinhardtii (ELDC94)

(72) A 35-pound container of solvent extracted crude canola oil was poured into large stainless-steel container and made uniform with IKA mixer.

(73) After mixing, approximately 1.5 kg of crude canola was placed into a 2 liter jacketed glass beaker with an overhead mixer with a square paddle and mixed at 90 revolutions per minute (rpm). The jacket temperature was set at 65? C. 0.7 grams of enzyme ELDC94 (reaction 1) or 7.5 grams of CHL26 (reaction 2), produced as described in Example 4, were added to the oil together with 100 grams of deionized water once the oil temperature had reached the set point. The material was shear mixed for 1 minute while covered with plastic wrap. The jacketed glass beaker was moved back to the overhead mixer and covered with plastic wrap. The materials were incubated with the enzymes for 24 hours at 250 rpm.

(74) 1.5 grams of 50% (wt. %) citric acid was added to the mixing oil. The set point of the jacket was reduced to 55? C. Once the material reached 55? C., the oil was moved to the shear mixer. 1.2 mL of 4 N NaOH was added to the oil and shear mixed 30 seconds. 0.3 grams of Purifine? Phospholipase C (PLC) and 30 grams of deionized water were added. The oil was shear mixed for 1 minute while covered with plastic wrap. The jacketed glass beaker was moved back to the overhead mixer and covered again with plastic wrap. The oil was mixed for 2 hours at 55? C. at 250 rpm.

(75) The beaker was moved back to the high shear mixer and 0.1 grams of a fungal phospholipase A.sub.1 (PLA.sub.1) enzyme was added to the oil and shear mixed 1 minute while covered with plastic wrap. The jacketed glass beaker was moved back to the overhead mixer and covered again with plastic wrap. The oil was mixed for 2 hours at 55? C. at 250 rpm. Increased the set point of the water bath to 75? C. Once the oil reached 75? C., the oil was centrifuged utilizing a Gyro-Centrifuge with the bowl with holes closed. Samples of the oil and gums were collected and analysed for the presence of P, Ca, Mg and Fe and chlorophyll derivatives (using HPLC) as described above.

(76) The mixture of oil and heavy phase remaining in the centrifuge bowl were poured in to a 400 mL beaker where the oil was decanted off. The remaining oil and heavy phase were placed into 50 mL centrifuge tubes and spun. The oil from the decanted bowl and in the tubes was discarded and liquid heavy phases were combined.

(77) The results in Table 6 and FIG. 4 a) show that the CHL26 enzyme having pyropheophytinase activity according SEQ ID NO: 1 is able to reduce chlorophyll derivatives in solvent extracted crude canola oil. Chlorophyll substrates are chlorophyll, pheophytin, and pyropheophytin and chlorophyll products are chlorophyllide, pheophorbide and pyropheophorbide.

(78) TABLE-US-00007 TABLE 6 Compounds (in ppm) in crude canola oil after treatment with enzymes CHL26 and the reference enzyme ELDC94 Chlorophyll derivatives (HPLC) (ppm) Enzyme P Ca Mg Fe Total Substrates Products None* 903.0 243.0 127 9.89 15.40 14.72 0.50 ELDC94 88.5 80.9 14.6 1.49 8.39 0.21 8.18 CHL26 82.0 77.3 14.1 1.58 9.15 1.26 7.89 *Starting material (crude canola oil)

(79) The results in FIG. 4 b) show that there are still unreacted phospholipids present in the collected heavy phase, which is an indication that the phospholipase reactions were too short to come to completion.

Example 7. Incubation of Pressed Crude Canola Oil with the CHL26 Enzyme at Varying Conditions

(80) A 35-pound container of pressed crude canola oil was poured into large stainless-steel container and made uniform with IKA mixer

(81) Reaction 3CHL26 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr, Followed by a 2 hr Incubation with PLA1

(82) About 1.5 kg of crude canola was placed into a 2 liter jacket glass beaker with an overhead mixer with a square paddle. The oil was mixed at 90 rpm. The jacket temperature was set at 70? C. 1.5 grams of 50% (wt. %) citric acid was added to the mixing oil and shear mixed 1 minute. The set point of the jacket was reduced to 60? C. Once the material reached 60? C., the oil was moved to the shear mixer. 1.2 mL of 4 N NaOH was added to the oil and shear mixed 30 seconds. 0.3 grams of Purifine PLC (LR79.14 Feb. 2018), 0.02 grams of Purifine PI-PLC, 7.5 grams of CHL26 enzyme [Hordeum vulgare var. distichum (barley, plant)], produced as described in Example 4, and 100 grams of deionized water. The material was shear mixed for 1 minute while covered with plastic wrap. The jacketed glass beaker was moved back to the overhead mixer and covered again with plastic wrap. The oil was mixed for 2 hours at 60? C. at 250 rpm.

(83) The jacketed glass beaker was again moved to the hear mixer where 0.075 grams of PLA.sub.1 (notebook, 0743B2) was added and the oil was shear mixed 1 minute. The jacketed glass beaker was moved back to the overhead and covered with plastic wrap. The oil was mixed and the reactions were allowed to continue for 2 hours at 250 rpm. Increased the set point of the water bath to 75? C. Once the oil reached 75? C., the oil was centrifuged utilizing Gyro-Centrifuge with the bowl with holes closed. Samples of the oil and gums were collected.

(84) The mixture of oil and heavy phase remaining in the centrifuge bowl were poured in to a 400 mL beaker where the oil was decanted off. The remaining oil and heavy phase were placed into 50 mL centrifuge tubes and spun. The oil from the decanted bowl and in the tubes was discarded and liquid heavy phases were combined.

(85) Reaction 4ELDC94 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr, Followed by a 2 hr Incubation with PLA.sub.1

(86) The same procedure from reaction 1 above was employed for enzyme ELDC94, using 0.61 grams of the formulated enzyme solution (produced as described in Example 4).

(87) Reaction 5CHL26 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr, Followed by a 4 hr Incubation with PLA.sub.1

(88) The same procedure was followed as reaction 1, but the PLA1 reaction was allowed to react for 4 hours instead of only 2 hours.

(89) Reaction 6CHL26 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr, Followed by a 4 hr Incubation with PLA.sub.1

(90) The same procedure was followed as reaction 3, except twice the amount of CHL26 (15 grams total) was added to the reaction.

(91) Reaction 7CHL26 Incubation with PLC and PI-PLC at Neutral pH for 2 hr, Followed by a 4 hr Incubation with PLA.sub.1

(92) The same procedure was followed as reaction 1, except no pH adjustment was made.

(93) Reaction 8SBO CHL26 Incubation with PLC and PI-PLC at pH 4.5 for 2 hr, Followed by a 2 hr Incubation with PLA.sub.1

(94) The same procedure as reaction 1, but the oil was a solvent extracted crude soybean oil (SBO).

(95) In Table 7 the phosphorus (P) and FIG. 5b), calcium (Ca), magnesium (Mg), and iron (Fe) contents of the oils and the respective gums before and after enzyme treatments according to reactions 1 to 6 are shown. At neutral pH, a higher amount of P remained in the oil as compared to reaction at pH 4.5.

(96) The results in Table 8 and FIG. 5a) show that the CHL26 enzyme converts a higher amount of chlorophyll derivatives in crude canola oil as compared to ELDC94, when the enzymes are incubated under the same conditions (reactions 1 and 2).

(97) In the present example the CHL26 enzyme converted a higher amount of chlorophyll substrates into the respective chlorophyll products in crude canola oil under neutral conditions as compared to acid conditions (pH 4.5) (compare reaction 7 with reactions 3, 5 and 6).

(98) The CHL26 enzymes also converts chlorophyll substrates in soybean oil into the respective chlorophyll products (reaction 8).

(99) The results in Table 8 also show that a higher amount of chlorophyll products were found in the gums (heavy phase) when the oil was reacted with the CHL26 enzyme as compared to the reaction with the ELDC94 enzyme.

(100) TABLE-US-00008 TABLE 7 Compounds in canola oil (Can) or soybean oil (SBO) after treatment with the CHL26 enzyme compared to reference enzyme ELDC94 and/or no enzyme treatment P Ca Mg Fe Reaction Oil pH (ppm) None, Crude Can 210 90.5 36.7 0.90 Rxn 3 - Can 4.5 10.7 7.9 1.7 0.20 CHL26 Rxn 4 - Can 4.5 4.4 3.3 0.9 0.07 ELDC94 None, Crude Can 210 90.5 36.7 0.90 Rxn 5 - Can 4.5 3.9 2.8 0.6 0.16 CHL26 Rxn 6 - Can 4.5 2.0 1.5 0.4 0.10 CHL26 Rxn 7- Can Neutral 103 80.9 10.5 0.88 CHL26 None, Crude SBO 773 66.2 64.3 0.76 Rxn 8 - SBO 4.5 5.8 0.5 0.7 0.04 CHL26

(101) TABLE-US-00009 TABLE 8 Chlorophyll derivatives in canola oil or soybean oil and the separated gums after treatment with the CHL26 enzyme compared to reference enzyme ELDC94 and/or no enzyme treatment Chlorophyll derivatives in Chlorophyll derivatives the oil (ppm) in the gums (ppm) Oil Substrates Products Substrates Products Crude Canola 13.13 0.90 Rxn 3 - CHL26, 4.19 7.97 0.06 6.39 pH 4.5 Rxn 4 - ELDC94, 10.28 2.62 0.18 3.06 pH 4.5 Crude Canola 13.13 0.90 Rxn 5, CHL26, 6.85 5.69 0.30 2.68 pH 4.5 Rxn 6 - CHL 26, 6.01 6.41 0.19 1.99 pH 4.5 Rxn 7 - CHL26, 1.26 10.02 b.d. 5.02 neutral pH Crude SBO 0.31 b.d. Rxn 8 - CHL26, 0.28 b.d. b.d. 0.56 pH 4.5 b.d.below detection,

Example 8. Use of CHL26 Enzyme in Caustic Refining Application of Canola Oil and Soybean Oil

(102) The following experiments are an evaluation of the CHL26 in a caustic refining application where the oil has been treated with a phosphoric acid and sodium hydroxide, as occurs in industrial processes of canola and soybean oils. A once refined product is an oil that was treated with phosphoric acid, then treated with sodium hydroxide to convert the Free Fatty Acids (FFA) into sodium soaps that are water soluble and removed in water or heavy phase of the refining centrifuge. The oil was then washed with water (2 to 10 percent w/w) to remove the remaining soaps and residual phospholipids present in the oil. Optionally, the enzymes were evaluated after the refining centrifuge in the water washing step, but at a much lower temperature.

(103) A five-gallon plastic pail of Once Refined Canola (ORCAN) oil was mixed with a high shear mixer to make uniform. 2-3 kg samples were pulled for use in the experiments below.

(104) Reaction 9ELDC94-Comparative

(105) 2 kg of once refined canola was placed into a 4 liter glass beaker on a hot plate with overhead mixing at 90 rpm. The oil was heated to 60? C. under agitation. Once the material reached 60? C., the beaker was moved to the shear mixer. 0.8 grams of enzyme ELDC94 (produced as described in Example 4) and 100 grams of deionized water were added to the oil. The material was shear mixed for 1 minute while covered with plastic wrap to minimize water loss. The glass beaker was moved back to the overhead mixer and covered with plastic wrap. The oil was mixed for 4 hours at 60? C. at 250 rpm. The temperature was increased to 75? C. The oil was centrifuged utilizing Gyro-Centrifuge. The separated oil was collected.

(106) The mixture of oil and heavy phase remaining in the centrifuge bowl were poured in to a 400 mL beaker where the oil was decanted off. The remaining oil and heavy phase were placed into 50 mL centrifuge tubes and spun. The oil from the decanted bowl and in the tubes was discarded and liquid heavy phases were combined. The heavy phase was a dark green.

(107) Reaction 10 ELDC94-Comparative

(108) Reaction 10 was a repeat of reaction 9, except 3 kg of oil was used and 2.0 grams of ELDC94 (produced as described in Example 4).

(109) After the analyses of the oils from reaction 9 and 10, the oils were combined mixed and analysed again.

(110) Reaction 11CHL26

(111) Reaction 11 was a repeat of reaction 9, except that 10.1 grams of CHL26 (produced as described in Example 4) was used instead of ELDC94. The heavy phase was a lighter green than reactions 9 and 10.

(112) Reaction 12CHL26

(113) Reaction 12 was a repeat of reaction 10, except that 20 grams of CHL26 was utilized.

(114) After analyses, the oils of reaction 11 and 12 were combined and mixed and after mixing analysed again.

(115) Reaction 13ELDC94-Comparative

(116) 3 kg of once refined soybean oil (ORSBO) was pulled from a caustic refining production line number 1 after the water washing centrifuge. The oil was placed into a 4 liter glass beaker and placed onto a hot plate with overhead mixing with a square mixing paddle (90 rpm). Once the material cooled 60? C., the beaker was moved to a shear mixer. 1.0 grams of ELDC94 enzyme produced as described in Example 4) and 150 grams of deionized water were added to the oil. The material was shear mixed for 1 minute while covered with a plastic wrap to minimize moisture loss. The glass beaker was moved back to the overhead mixer and again covered with a plastic wrap. The oil was mixed for 4 hours at 60? C. at 250 rpm. The temperature was increased to 75? C. and then the oil was centrifuged utilizing Gyro-Centrifuge.

(117) Collected oil and heavy samples for further analyses.

(118) The remaining oil and heavy phase remaining in the centrifuge bowl were poured in to a 400 mL beaker where the oil was decanted off. The remaining oil and heavy phase were placed into 50 mL centrifuge tubes and spun. The remaining oil in the tubes was discarded and liquid heavy phases were combined. The heavy phase was colorless, no discernible color pigments.

(119) Reaction 14CHL26

(120) Reaction 14 was a repeat of reaction 13, except that 15 grams of CHL26 (produced as described in Example 4) was utilized instead of ELDC94.

(121) Reaction 15EDLC94-Comparative

(122) 3 kg grams of once refined soybean oil (ORSBO) was pulled from a caustic refining production line number 1 after the water washing centrifuge. The oil was placed into a 4 liter glass beaker and placed onto a hot plate with overhead mixing with a square mixing paddle (90 rpm). Once the material cooled 60? C., the beaker was moved to a shear mixer. 1.2 grams of ELDC94 enzyme produced as described in Example 4 and 150 grams of deionized water were added to the oil. The material was shear mixed for 1 minute while covered with a plastic wrap to minimize moisture loss. The glass beaker was moved back to the overhead mixer and again covered with a plastic wrap. The oil was mixed for 4 hours at 60? C. at 250 rpm. The temperature was increased to 75? C. and then the oil was centrifuged utilizing Gyro-Centrifuge.

(123) Collected oil and heavy phase (gums) samples for further analyses.

(124) The remaining oil and heavy phase remaining in the centrifuge bowl were poured in to a 400 mL beaker where the oil was decanted off. The remaining oil and heavy phase were placed into 50 mL centrifuge tubes and spun. The remaining oil in the tubes was discarded and liquid heavy phases were combined. The heavy phase was colorless, no discernible color pigments.

(125) Reaction 16CHL26

(126) Reaction 16 was a repeat of reaction 15, except that 15 grams of CHL26 was utilized.

(127) The results of reactions 9 to 16 and the results of the combined and mixed oils from reaction 9 and 10 and from reactions 11 and 12 are shown in Table 9 and FIG. 6.

(128) The results in Table 9 and FIG. 6 show that the enzyme CHL26, having pyropheophytinase converts a higher amount of chlorophyll substrates (chlorophyll, pheophytin and pyropheophytin) to its chlorophyll products (chlorophyllide, pheophorbide, pyropheophorbide) than the reference chlorophyllase enzyme ELDC94.

(129) TABLE-US-00010 TABLE 9 Chlorophyll derivatives (substrates and products) in once refined canola oil (ORCAN) and once refined soybean oil (ORSBO) after caustic refining and after treatment with the CHL26 enzyme and the ELDC94 (reference) enzyme Chlorophyll derivatives in oil (ppm) Enzyme reaction Substrates Products None: ORCAN 27.38 b.d. Rxn 9 - ELDC94 7.87 4.99 Rxn 10 - ELDC94 19.37 0.42 Combined 9 & 10 18.71 0.39 Rxn 11 - CHL26 11.60 4.96 Rxn 12 - CHL26 n.m. n.m. Combined 11 & 12 12.00 6.19 None: ORSBO 3.85 b.d. Rxn 13 - ELDC94 1.09 0.06 None: ORSBO 3.90 b.d. Rxn 14 - CHL26 1.12 0.18 None: ORSBO 3.90 b.d. Rxn 15 - ELDC94 2.05 b.d. Rxn 16 - CHL26 1.77 0.07 b.d. = below detection n.m. = not measured

(130) The results of Table 10 show the contents of free fatty acids (FFA), soap and phosphor and Ca, Mg in once refined canola oil and once refined soybean oil after enzymatic treatments described above.

(131) TABLE-US-00011 TABLE 10 Composition of once refined canola oil (ORCAN), once refined soybean (ORSBO) oil after caustic refining and after treatment with the CHL26 enzyme and the ELDC94 (reference) enzyme FFA Soap P Ca Mg Fe (%) (ppm) (ppm) (ppm) (ppm) (ppm) ORCAN 0.05 195 4.5 0.9 0.2 0.03 Rxn 9 - 0.05 b.d. 0.5 0.7 tr 0.02 ELDC94 Rxn 10 - 0.07 b.d. 0.6 1.8 tr 0.03 ELDC94 Combined 0.06 b.d. 0.6 0.7 tr 0.07 9 & 10 Rxn 11 - 0.06 tr 1.6 2.4 0.1 0.11 CHL26 Rxn 12 - 0.06 b.d. 1.7 2.9 0.1 0.07 CHL26 Combined 0.06 tr 1.7 2.7 0.1 0.09 11 & 12 ORSBO 0.12 20 0.3 0.2 b.d. b.d. Rxn 13 - 0.10 b.d. 0.2 0.1 b.d. b.d. ELDC94 ORSBO 0.06 27 1.0 0.4 tr b.d. Rxn 14 - 0.05 b.d. 0.2 0.1 b.d. b.d. CHL26 ORSBO 0.03 242 2.8 0.7 0.2 tr Rxn 15 - 0.02 tr 0.3 0.2 b.d. 0.1 ELDC94 ORSBO 0.05 396 3.3 0.9 0.2 b.d. Rxn 16 - 0.03 tr b.d. 0.2 b.d. b.d. CHL26 tr = trace b.d. = below detection n.m. = not measured