WO2008074083A1 - Extracting and purifying lipoxygenase - Google Patents
Extracting and purifying lipoxygenase Download PDFInfo
- Publication number
- WO2008074083A1 WO2008074083A1 PCT/AU2007/001971 AU2007001971W WO2008074083A1 WO 2008074083 A1 WO2008074083 A1 WO 2008074083A1 AU 2007001971 W AU2007001971 W AU 2007001971W WO 2008074083 A1 WO2008074083 A1 WO 2008074083A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- extract
- lipoxygenase
- solution
- plant matter
- process according
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
Definitions
- the present invention relates to the extraction and purification of lipoxygenase from plant matter, including soy beans.
- Lipoxygenase also known as lipoxidase, lipoperoxidase, and carotene oxidase (EC1.13.11.12) catalyse the oxidation of unsaturated fatty acids in lipids containing cis- 1-4-pentadiene system by molecular oxygen to hydroperoxides. Linolenic and linoleic acids are the most common fatty acid moieties in unsaturated lipids that act as substrates. Accordingly, one reaction catalysed by lipoxygenase is:
- Lipoxygenase also oxidises other methylene-interrupted polyunsaturated fatty acids.
- Lipoxygenase is found in plants including soy beans and other beans such as navy beans, kidney beans and lima beans. It is also found in common legumes i.e. beans, peas, lentils, etc. It is also found in potato, horse radish, turnips, and other tubers. Various types of seeds, bulbs and nuts also contain lipoxygenase.
- LOX1 Three lipoxygenase isozymes, LOX1 , LOX2, and LOX3 (accession no.s P08170, P09439 and P09186 respectively) have been found in soy bean cotyledons. Other forms, LOX4 (P38417) and LOXX (P24095), have also been observed.
- Lipoxygenase as found in soy flour is used in the baking industry to improve dough and crumb quality and to bleach pigments in the dough during breadmaking. In these applications, lipoxygenase reduces the binding of added shortening with protein during dough mixing, increases gluten strength, and improves baking performance and product quality.
- the lipoxygenase is provided to the dough in the form of enzyme active soy flour. It is destroyed during the baking process by thermally-induced denaturation.
- a number of groups have sought to purify lipoxygenase. Each of these groups has taken the approach of using de-fatted soy bean flour as a starting material.
- An aqueous suspension is prepared in the form of a slurry. After removing solids from the solution, ammonium sulphate is added to precipitate proteins and hence to concentrate lipoxygenase. The ammonium sulphate must then be removed from the concentrated lipoxygenase-protein matrix. In some circumstances, this removal of ammonium sulphate requires dialysis. In other circumstances, more specialised chromatography is required. Consequently, many processes for purifying lipoxygenase from ancillary proteins have limited commercial application.
- the invention seeks to minimise or reduce the above limitations or problems and in certain embodiments to provide a process for producing a purified extract of a lipoxygenase enzyme from a plant matter.
- the process includes the steps of:
- the inventor has found that by using plant matter as a starting material for the purification of a lipoxygenase, a much greater yield of lipoxygenase can be obtained.
- a yield of 3 to 4 times more enzyme could be obtained from soy beans extracted according to the process described herein as compared with enzyme obtained from an extraction involving enzyme active soy flour.
- a total purification factor approaching 125 fold was obtained when the extract obtained by the process described herein was further subjected to ion exchange chromatography and ultrafiltration.
- One particular advantage is that according to the process described herein, the specific activity of the lipoxygenase can be increased and hence the enzyme enriched in the extract without having to use an ammonium sulphate concentration step.
- lipoxygenase is extracted from the plant matter into the aqueous solution.
- Water is one example of an aqueous solution.
- Other solutions for example salt solutions or pH adjusted solutions that facilitate the transfer of the lipoxygenase from the plant matter into the solution may also be used.
- the transfer of the lipoxygenase into the solution is facilitated by applying a chemical or physical treatment to the plant matter that facilitates ingress of the aqueous solution into the plant matter. With this treatment, the tissue of the matter is made vulnerable or otherwise exposed to the solution. Accordingly, tissue that would otherwise be less readily hydrated by the solution becomes hydrated, resulting in an increase in the amount of enzyme that may be extracted from the plant matter.
- Other physical treatments known to the skilled worker may also be applied. Whether these are applicable will depend largely on the physical characteristics of the plant matter to be treated and taking account one objective of the first step of the process which is to extract as much enzyme from the plant matter into the solution as is required by the particular application.
- the extract is formed from milling the plant matter to facilitate ingress of the aqueous solution into the plant matter. This produces a hydrated slurry.
- One way of facilitating transfer of the enzyme into the solution is to steep the plant matter in the aqueous solution to form a steep solution.
- the amount of time required for steeping depends again on the physical characteristics of the plant matter to be treated and the need to extract as much enzyme from the plant matter into the solution as is required by the particular application. Steeping times may be as little as 1 hour for some plant matter. Where the plant matter is a bean or legume, the steeping time is about 6 - 30 hours. Longer times may be used although this may require providing conditions to prevent microbial contamination or spoilage.
- the solution at the completion of the steeping step, the solution has generally absorbed or seeped or soaked into the plant matter leaving it generally hydrated throughout.
- the plant matter is steeped in the aqueous solution to form a steep solution, before the plant matter is milled.
- the inventor has found that the amount of enzyme extracted from the plant matter can be substantially increased when a subsequent physical treatment described above is applied to the steeped plant matter in the presence of the steep solution.
- One advantage is that the extraction efficiency of the process may be increased.
- Another is that the production costs are decreased by minimising water usage.
- the plant matter is milled in the steep solution.
- the aqueous solution may be provided with an alkaline pH, for example a pH of about 7 to 9.
- an alkaline pH for example a pH of about 7 to 9.
- the plant material may be maintained in the aqueous solution for about 1 to 30 hours. Shorter or longer times are possible.
- the extract is provided with an alkaline pH and milled to facilitate extraction of the lipoxygenase into the solution.
- particulate matter in the form of cellular or tissue debris may become dispersed or otherwise contaminate the solution.
- the process includes filtering the solution to remove cellular material from the solution, to form the extract.
- the process includes heating and reducing the pH of the solution to separate compounds in the solution from lipoxygenase, to form the extract.
- the process of the invention includes forming an acidified extract from the extract formed in the first step of the process.
- One objective of this step is to selectively purify the lipoxygenase in solution from other compounds, especially proteins in the solution by causing the latter to aggregate or to flocculate and so to precipitate out of solution, leaving the lipoxygenase in solution.
- This step is based on the phenomenon known as "isoelectric precipitation” or "fractional precipitation” wherein a protein species is observed to fall out of solution at a given pH (its isoelectric point) at which it has no net charge.
- the isoelectric point of LOX1 is about 5.8, LOX2 about 6.2 and LOX3 about 6.3.
- the pH of the extract is adjusted to about pH 4.5 to form the acidified extract.
- higher or lower pH conditions could be applied depending on the isozyme to be purified and the isoelectric point or "pi" of the contaminant proteins in the extract prior to acidification.
- the extract is heated, for example to about 55 0 C as it is acidified to pH 4.5 to form the acidified extract.
- the pH of the solution may be ramped down in a step wise manner to gradually cause precipitation of contaminant proteins using citric acid or glucono delta lactone or "GDL".
- the precipitated proteins may be separated from the lipoxygenase in the acidified extract by a number of approaches.
- the acidified extract is allowed to cool, or otherwise chill, and the precipitated protein allowed to settle, after which the acidified extract is removed, for example, by siphoning, to separate the extract from the precipitated protein.
- the acidified extract is chilled to facilitate separation of the precipitated compounds from the extract, hence providing the purified extract.
- the flocculated protein is separated by a filtration process at 55 0 C as an enhancement to chilling to improve purification of the enzyme. This process provides for a large agglomeration of proteins more easily separated by filtration.
- the pH of the acidified extract may be adjusted back to alkaline conditions, for example, to about pH 9 to provide an enhanced activity of the enzyme.
- the extract may then be further processed to concentrate the lipoxygenase, selectively removing solvent from the purified extract to increase the concentration of the lipoxygenase in the purified extract.
- This can be done by a number of approaches including precipitation and re-dissolution, chromatography etc.
- the inventor has found microfiltration, ion exchange and ultrafiltration to be a particularly useful approach.
- the process includes selectively removing solvent from the purified extract to isolate and increase the concentration of the lipoxygenase in the purified extract.
- Example 2 Activity of lipoxygenase obtained from sov flour as compared with soy bean.
- Full fat enzyme active soy flour was prepared as a 1:5 w/v suspension at pH 9.0 (0.1 M Tris HCL) with 0.1% w/v surfactant (Triton-X-100) and processed for 6 hours. The enzyme activity at this time was found to be 45 units.
- Soy beans by comparison were prepared as a 1:5 w/v suspension and soaked at pH 9.0 (0.1 M Tris HCL) with 0.1% w/v surfactant (Triton-X-100) for 6-24 hours and processed using a colloid mill incorporating the steep water during the milling process.
- Tris HCL 0.1 M Tris HCL
- Triton-X-100 0.1% w/v surfactant
- One unit of lipoxygenase activity is the amount of enzyme required to decrease the absorbance of methylene blue in the defined assay conditions by 0.001 absorbance units at 660 nm in 1 minute at 25 0 C.
- the pH of supernatant is decreased to 7.0 to prepare the extract for heating
- the supernatant at pH 7.0 is heated to 55 0 C and held for 15 min to isolate lipoxygenase from ancillary proteins
- the extract is then filtered at 55 0 C through a 40 ⁇ m screen to remove the flocculated protein.
- the pH of the solution is adjusted back to pH 9.0
- the filtrate is cooled in either a heat exchanger or is left to settle for 6-24 hours to clarify the extract
- Centrifugation/Filtration may be used to remove any secondary protein flocculation
- Clarified extract is loaded on to DEAE column with 25 mM Tris- HCL, pH 7 with 0.5 M NaCI as the eluent
- the beans are removed from the buffer.
- the beans and buffer are then fed into the colloid mill at a ratio of 1 :4 (beans to buffer) at a flow rate of 3L/min buffer. There needs to be a steady flow of liquid into the colloid mill to minimise heat build up in the extract.
- pH of the milled extract is maintained at pH 9.0 to maximise extraction from plant material (also this is optimum pH for LOX1 activity).
- the macerated soy bean extract can be further held at this pH for 1 hour to improve extraction of LOX1 from the bean extract.
- the crude extract is passed through a 3 tiered vibrating screen using a diaphragm pump.
- the flow rate is controlled so there is no excess build up of unwanted cellular material on the 500 ⁇ m top screen.
- the three mesh screens that the extract passes through are 500 ⁇ m, 250 ⁇ m and IOO ⁇ m.
- Cellular material is removed by the vibration of the screen that permits liquid to pass through the screen while solid material above the cut off is retained and removed by the solids spout. Once the liquid is passed through the vibrating screen, it is collected and transferred to a mixer for the heating and acidification step. pH is adjusted to pH 7.0 for the heating step.
- the parameters for heating are set at 55 0 C. Once this temperature is achieved, the extract is maintained at this temperature for 15 min, the extract is then acidified to pH 4.5 using 1 M citric acid. Alternatively, known amounts of freshly made GDL solution can be mixed with the extract prior to heating with the pH drop due to conversion of GDL to gluconic acid is monitored.
- the chilled extract is either passed through a heat exchanger or transferred to a conical tank and held for 24 hours in a cold room at ⁇ 10°C for settling of the soy protein isolate. After the settling has occurred the clarified extract is siphoned from the top and removed. The bottom half of the extract is filtered through a 40 ⁇ m mesh screen then centrifuged to remove unwanted protein.
- the clarified extract is then adjusted to pH 9.0 for further filtration while providing maximum enzyme activity
- the extract is microfiltered through a 0.45 ⁇ m membrane and the permeate collected.
- Ion exchange Lipoxygenase may be isolated by DEAE anionic exchange chromatography using 25 mM Tris-HCL (pH 7.0) at a flow rate of 3 mL per minute on a 50 mLI column and eluted with 0.5 M sodium chloride.
- the extract is concentrated using a IO kDa NMWC (concentrated 10-30 fold its original volume).
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- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
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- General Engineering & Computer Science (AREA)
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- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/520,745 US20100144009A1 (en) | 2006-12-20 | 2007-12-20 | Extracting And Purifying Lipoxygenase |
NZ578368A NZ578368A (en) | 2006-12-20 | 2007-12-20 | Extraction and purification of lipoxygenase from soy beans |
JP2009541692A JP2010512757A (en) | 2006-12-20 | 2007-12-20 | Extraction and purification of lipoxygenase |
EP07845411A EP2118126A4 (en) | 2006-12-20 | 2007-12-20 | Extracting and purifying lipoxygenase |
AU2007335258A AU2007335258B2 (en) | 2006-12-20 | 2007-12-20 | Extracting and purifying lipoxygenase |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2006907091A AU2006907091A0 (en) | 2006-12-20 | Extracting and purifying lipoxygenase | |
AU2006907091 | 2006-12-20 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2008074083A1 true WO2008074083A1 (en) | 2008-06-26 |
Family
ID=39535891
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/AU2007/001971 WO2008074083A1 (en) | 2006-12-20 | 2007-12-20 | Extracting and purifying lipoxygenase |
Country Status (6)
Country | Link |
---|---|
US (1) | US20100144009A1 (en) |
EP (1) | EP2118126A4 (en) |
JP (1) | JP2010512757A (en) |
AU (1) | AU2007335258B2 (en) |
NZ (1) | NZ578368A (en) |
WO (1) | WO2008074083A1 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5887714B2 (en) * | 2010-06-07 | 2016-03-16 | 不二製油株式会社 | Soy emulsified composition, reduced-fat soy milk, and production method thereof |
US8293790B2 (en) | 2011-10-19 | 2012-10-23 | Dignity Sciences Limited | Pharmaceutical compositions comprising DGLA and benzoyl peroxide and methods of use thereof |
CN106170470B (en) * | 2013-10-29 | 2019-06-18 | 尊贵科学有限公司 | The method for manufacturing 15- hydroxy fatty acid derivative |
CN109852591B (en) * | 2019-01-20 | 2022-11-08 | 齐鲁工业大学 | Method for extracting lipoxygenase from soybean whey wastewater by reverse pH gradient |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6627797B1 (en) * | 2000-03-21 | 2003-09-30 | The Texas A&M University System | Maize lipoxygenase polynucleotide and methods of use |
DE60138201D1 (en) * | 2000-10-02 | 2009-05-14 | Fuji Oil Co Ltd | FRACTIONIZED SOYBEAN PROTEIN AND METHOD FOR THE PRODUCTION THEREOF |
-
2007
- 2007-12-20 NZ NZ578368A patent/NZ578368A/en not_active IP Right Cessation
- 2007-12-20 EP EP07845411A patent/EP2118126A4/en not_active Withdrawn
- 2007-12-20 JP JP2009541692A patent/JP2010512757A/en not_active Ceased
- 2007-12-20 US US12/520,745 patent/US20100144009A1/en not_active Abandoned
- 2007-12-20 WO PCT/AU2007/001971 patent/WO2008074083A1/en active Application Filing
- 2007-12-20 AU AU2007335258A patent/AU2007335258B2/en not_active Ceased
Non-Patent Citations (6)
Title |
---|
BISAKOWSKI B. ET AL.: "Characterization of Purified Lipoxygenase Extracts from Fusarium Proliferatum", J. AGRIC. FOOD CHEM., vol. 46, 1998, pages 2382 - 2388, XP008112429 * |
BISAKOWSKI B. ET AL.: "Partial Characterization of a Lipoxygenase from Fusarium proliferatum", FOOD BIOTECHNOLOGY, vol. 9, no. 3, 1995, pages 189 - 205, XP008112811 * |
NICOLAS J. ET AL.: "Purification and Some Properties of Wheat Germ Lipoxygenase", JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, vol. 33, 1982, pages 365 - 372, XP008111646 * |
PERRAUD X. ET AL.: "Characterization of a Lipoxygenase Extract from Penicillium sp", J. OF THE AMER. OIL CHEMISTS'SOC, vol. 77, no. 4, 2000, pages 335 - 342, XP008111680 * |
See also references of EP2118126A4 * |
YABUUCHI S. ET AL.: "Partial Purification and Characterization of the Lipoxygenase from Grains of Hordeum Distichum", PHYTOCHEMISTRY, vol. 14, 1975, pages 2569 - 2572, XP008112430 * |
Also Published As
Publication number | Publication date |
---|---|
EP2118126A1 (en) | 2009-11-18 |
EP2118126A4 (en) | 2010-02-03 |
NZ578368A (en) | 2011-02-25 |
US20100144009A1 (en) | 2010-06-10 |
JP2010512757A (en) | 2010-04-30 |
AU2007335258B2 (en) | 2012-12-20 |
AU2007335258A1 (en) | 2008-06-26 |
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