WO1982004066A1 - Process for isolation of proteins from plant leaves - Google Patents

Process for isolation of proteins from plant leaves

Info

Publication number
WO1982004066A1
WO1982004066A1 PCT/US1981/000655 US8100655W WO8204066A1 WO 1982004066 A1 WO1982004066 A1 WO 1982004066A1 US 8100655 W US8100655 W US 8100655W WO 8204066 A1 WO8204066 A1 WO 8204066A1
Authority
WO
WIPO (PCT)
Prior art keywords
process according
liquid portion
ribulose
proteins
diphosphate carboxylase
Prior art date
Application number
PCT/US1981/000655
Other languages
English (en)
French (fr)
Inventor
Proteins Inc Leaf
Samuel G Wildman
Prachuab Kwanyuen
Original Assignee
Leaf Protein Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Leaf Protein Inc filed Critical Leaf Protein Inc
Priority to BR8109020A priority Critical patent/BR8109020A/pt
Priority to DE3152847T priority patent/DE3152847C2/de
Priority to GB08300606A priority patent/GB2110221B/en
Priority to PCT/US1981/000655 priority patent/WO1982004066A1/en
Priority to JP50228781A priority patent/JPS58500737A/ja
Publication of WO1982004066A1 publication Critical patent/WO1982004066A1/en

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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
    • C12N9/88—Lyases (4.)

Definitions

  • this invention relates to a process for isolating proteins from plant leaves. In another aspect it relates to a process for obtaining ribulose 1, 5-diphosphate carboxylase from the green leaves of plants. In another, and more specific, aspect it relates to a process for obtaining ribulose 1,5- diphosphate carboxylase from tobacco leaves.
  • the succulent leaves of certain plants are composed of 10-20% solids, the balance being water.
  • the solid portion is composed of a water- soluble portion and a water-insoluble portion, the latter being made up, for the most part, of the fibrous struc ⁇ tural material of the leaf.
  • the water-soluble compounds are divisible into two groups.
  • One group includes compounds of relatively lower molecular weight such as sugars, vitamins, amino acids and other compounds whose molecular weight do not exceed about 10,000.
  • the second group is almost exclusively proteins whose molecular weights are about 30,000 or greater.
  • the proteins can be resolved into two fractions.
  • One fraction contains a mixture of proteins whose molec- -2- ular weights range from about 30,000 to 100,000. These proteins are sometimes referred to as "Fraction 2 pro ⁇ teins.”
  • the remaining fraction comprises a single protein having a molecular weight of about 550,000 and is sometimes referred to as "Fraction 1 protein.”
  • Fraction 1 protein was first identified in 1947. Subsequent research lead to the discovery that this protein was an enzyme involved in photosynthesis. Since then, it has been given a number of names. Among these are ribulose, 1,5-diphosphate carboxylase, carboxydis- mutase, ribulose 1,5-bisphosphate carboxylase and ribu ⁇ lose 1,5-di (or bis) phosphate carboxylase-oxygenase. Fraction 1 protein may compose up to 25% of the total protein content of a leaf and up to 10% of the solid matter in the leaf. In 1970 it was discovered that crystalline Fraction 1 protein could be obtained from tobacco leaves.
  • Fraction 1 protein when pure, is odorless, taste ⁇ less and colorless and has high nutritional value. In view of these properties, and because it can be obtained in high purity. Fraction 1 protein is considered to have a potentially valuable application as a food supplement for animals and humans. In the case of humans, the additive could be a component of high protein or other special diets. It has, for example, been suggested as a supplement to the diet of persons who require dialysis because of kidney disease.
  • Fraction 1 protein is not a commercially important prod- uct since the processes known to the art for obtaining it from vegetable matter are not commercially feasible.
  • Each published method begins with pulping the leaves, or leaves and stalk of the plant, followed by expressing a green juice from the pulp.
  • the green juice which contains finely particulate green pigmented material.
  • ⁇ ' -3- is clarified, for example, by filtration or centrifuga- tion, to separate the finely particulate solid matter from the liquid.
  • the resulting liquid is brown in color.
  • the first method described for isolating Fraction 1 protein involved concentration of Fraction 1 protein simultaneously with its partial separation from lower molecular weight compounds in the brown juice by molec ⁇ ular filtration. Using a molecular sieve whose pores would pass smaller molecules without passing Fraction 1 protien, the brown juice was placed under pressure so that small molecules would pass through the pores. The solution containing the Fraction 1 protein was concentrated about ten-fold and the dialyzed to remove additional small molecules in the solution. Dialysis was accomplished using a collodion-type dialysis bag.
  • the pores of the bag would not permit passage of the Fraction 1 protein but allowed the smaller molecules to escape through the bag into water.
  • crystals of Fraction 1 protein formed.
  • the second method developed to isolate the Fraction 1 protein involved passing the brown juice obtained from the leaves through a Sephadex chro atographic column.
  • Sephadex consists of water insoluble microscopic beads of polymerized sugar. Either Sephadex G-25 or G-50 was used to perform the separation. Selection of proper beads permits small molecules to penetrate to the interior of their structure to the exclusion of larger molecules. The larger molecules, therefore, are only found in the liquid in the inter- stices between the tightly packed Sephadex beads. This interstitial space is referred to as the "void volume".
  • the volume of brown juice cannot exceed about 25% of the total volume of the beads.
  • the beads are first equilibrated with a buffer and a volume of brown juice, containing the same buffer, is then layered on top of the Sephadex column.
  • the brown liquid is eluted from the column using the
  • Fraction 1 protein does not crystal- lize, as is the case with the extract of all plants except tobacco, ammonium sulfate is added until the solution is 30-50% saturated. This leads to precipita ⁇ tion of an amorphous material which is collected by centrifugation. After separation, the precipitate is redissolved in a smaller volume of buffer than that from which it was precipitated to which is added 8% polyethyl ⁇ ene glycol. This mixture is placed in an open dish adjacent to another open dish containing silica gel and the two dishes confined in a closed vessel. Water is gradually evaporated from the protein solution and absorbed by the silica gel. With the passage of time, crystals of Fraction 1 protein develop.
  • ribulose 1,5- diphosphate carboxylase is obtained in crystalline form by adjusting the pH of the liquid portion of a plup derived from the leaves to a value in the range of from between about 6 to a pH above that at which the pro- tein will denature and precipitate as an amorphous mass, i.e., to a value above the isoelectric point which occurs at about pH 5.0.
  • the liquid after separation of insol ⁇ uble material, is then permitted to stand, preferably while cooled below ambient temperature, to permit crys- tallization of the Fraction 1 protein. It has been our general observation that crystallization occurs more readily as the pH decreases, i.e., nears the isoelectric point.
  • An object of this invention is to provide an i - proved process for the isolation of Fraction 1 protein. Another object is to obtain Fraction 1 protein in high yield and purity.
  • the process of the present invention may be used to isolate ribulose 1,5-diphosphate carboxylase and lower molecular weight protein from leaves of many varieties.
  • tobacco leaves, and particularly the leaves of immature tobacco are a particularly rich source of the protein, it is preferred to use such leaves in the process of the invention. Accordingly, the invention will be described with specific _reference to the isolation of ribulose 1,5-diphosphate carboxylase from tobacco.
  • the pulping process is carried out in the presence of a reducing agent.
  • the pulping process permits phenol oxidase enzymes present in the leaves to contact the leaf proteins. This results in oxidation of aromatic amino acids such as tyrosine which comprise part of the primary structure of proteins. This oxida ⁇ tion modifies the protein, made visually manifest by their becoming brown, and lowers their solubility in water.
  • the reducing agent in effect, acts as an anti- oxidant to suppress this oxidation.
  • the presently preferred reducing agent for use in the invention is 2-merca ⁇ toethanol because it is volatile and evaporates during the further processing described below leaving little or no residue in the material isolated.
  • other reducing agents may also be used. Among these are agents such as sodium metabisul- fite and dithiothreitol. Separation of the residue of these agents, if any, can be done using conventional techniques.
  • the amount of reducing agent sufficient to control the oxidation can vary depending, for example, on the agent selected. In the case of 2-mercaptoethanol, effective suppression of the undesirable oxidation can be achieved using about 5 milliliters of the liquid agent per kilogram of plant material being processed.
  • the liquid portion of the plant material contains the plant proteins, including Fraction 1, and other
  • the solid portion of the pulp includes coarse, easily separated material and finely particulate green pigmented material which is difficult to separate from the liquid.
  • the coarse , material is preferably separated from the liquid portion promptly after the leaf material is converted to a pulp. A simple filtration, for example, using cheese cloth, will accom ⁇ plish this separation.
  • the liquid portion which still contains the finely particulate, green pigmented material is treated with acid, preferably hydrochloric acid, as necessary, to bring the pH to within the desired range, i.e., to within the range from about pH 6.0 to a point near but above the isoelectric point at which the protein in the liquid portion is denatured whereby it precipitates as an amorphous mass.
  • acid preferably hydrochloric acid
  • This mass contains both Fraction 1 and Fraction 2 protein material.
  • the isoelectric point of proteins is at about pH 5.0. Therefore, the practical lower limit to which the pH should be adjusted according to the present invention is about pH 5.3. Separation of the coarse material may be carried out after acidification of the liquid.
  • Other mineral acids, including phosphoric and sulfuric may also be used.
  • the pH of the liquid portion of tobacco leaf pulp varies according to the age of the plant. In the case of very young plants, i.e., plants less than about 12" tall, the pH will be in the range of about 6.0 or higher. As the plants mature, the pH of the liquid portion decreases, i.e., the liquid portion is naturally more acidic. For example, the pH of liquid from plants in the range of from 18" to 24" in height was about pH 5.7, whereas the liquid portion derived from plants 24" to 36" had a pH of about 5.3.
  • the pH of the liquid portion is adjusted to a range from about 5.4 to 5.8, most preferably to a pH of about 5.4-5.6.
  • the pH is above about 5.8, crystallization occurs at a signifi- j fREA
  • the heating step has the effect of coagulating the finely particulate material to an extent that permits its separation by centrifuging as discussed above.
  • the entire pulp may be heated to facilitate separa ⁇ tion of the green finely particulate material.
  • the heating step must be carried out at a tempera- ture below that at which the protein will denature by heat alone. Generally, therefore, the heating step should be carried out below about 52° C as heating about that temperature results in precipitation of the protein.
  • the heat treatment can be performed either as a continuous or batch process as described in our copending application Serial No. 78,505. Thus, in a batch process, the pulp is placed in a vessel whereby he .
  • the pulp is trans- ferred to the pulp under conditions where no part of the > pulp, or at ' least the liquid portion thereof, is heated to a temperature at which the protein denatures.
  • a temperature at which the protein denatures As indicated above, preferably the pulp is heated to a temperature of 50° C • _ 1° for from about 15 minutes to about 20 minutes.
  • the pulp is pumped without undue agitation through coils immersed in a liquid heat to a temperature such that, by heat exchange, a specified volume of pulp would be heated to 50° C -_ 1° for from about 15 minutes to about 20 minutes and then through coils in contact with liquid at a temperature lower than 50° C to reduce the temperature of the pulp.
  • Heating the pulp as described above to cause coagu ⁇ lation of the finely particulate material is more effi- cient than merely adjusting the pH.
  • the heating regimen should be used only if necessary to remove the finely particulate material since it has the actual effecting of increasing the time over which crystalliza ⁇ tion of the Fraction 1 protein occurs and, in some caes, the amount of protein obtained.
  • the finely particulate material can be separated after coagulation, for example, by centrifuga ⁇ tion, from the liquid portion.
  • the supernatant liquid obtained is a brown juice.
  • This juice is stored at a temperature at which crystallization will occur, ususally at or below room temperature to obtain crystal of Frac ⁇ tion 1 protein.
  • the maximum storage time required is not more than about 16 hours. It has been our experience that storage beyond 16 hours does not usually improve yields.
  • the crystallized ribulose 1,5-diphosphate carboxylase is separated from the supernatant liquid by filtration or centrifugation (3000 RPM, 5 rninw ).
  • the crystalline form of " ribulose 1,5-diphosphate carboxylase differs from the three forms obtained " using prior art processes.
  • Form I crystals have the shape of dodecahedrons and are produced by the previously men ⁇ tioned processes using molecular sieves or Sephadex chromatography.
  • Form II crystals have the shape of extremely thin plates and so far have been produced only under very special conditions and in extremely small amounts for x-ray crystallographic studies.
  • Form III crystals are tetragonal bipyramids and are produced by the ammonium sulfate and polyethylene glycol treatment previously mentioned.
  • the Fraction 1 protein crystals pro ⁇ quizd by the process described herein take yet a fourth form, unlike the other three forms of crystals known to the art.
  • Fraction 1 protein crystals are obtained having an apparent octagonal form, by microscopic examination, in high yield and in high purity.
  • Fraction 1 protein may be removed from the supernatant by acidifying it to at or below the isoelectric point, i.e., to a pH of 5.0 or below. This causes the proteins in solution to precipi ⁇ tate. Highest yields of protein are obtained by adjust ⁇ ing the pH to about 4.0-4.5 by the addition of hydro ⁇ chloric acid or other suitable acids. The resulting precipitate can be collected by centrifugation (3000 RPM, 5 min.). Contaminants can be removed if the precipitate is washed with water and again collected by centrifuga ⁇ tion.
  • Type NC95 tobacco plants are cultivated at a plant density of 0.5 square feet per plant until a height of 18-24 inches is attained.
  • the plants are cultivated in such a way that the leaves are deep green in color.
  • the entire aerial portions of the plants are harvested and cut into pieces small enough to be introduced into a one-gallon size Waring blender.
  • the blades of the blender are covered with about 200 ml. of water. " (The Waring blender will not disintegrate the plant material unless the ' blades are submerged in a liquid. However, with other such devices, such as a Rietz disintegrator, addition of water would be unnecessary. )
  • a one-kilogram batch of coarsely chopped stems and leaves obtained from the harvested plant material is added to the water with 5 ml. of 2-mercaptoethanol and blended to a smooth pulp.
  • the resulting pulp which has the consistency of a thick pancake batter, consists of a volume of about 1.2 liters.
  • the coarse material in the pulp was poured onto two layers of 24/20-mesh cheese cloth supported on an 8-inch diameter, 32-mesh sieve which is placed in a large funnel draining into a col ⁇ lecting flask.
  • the 1.2 liters of pulp yields approximately 1.0 liter of liquid containing green pigmented material.
  • the "green juice” had a pH of from about 5.7 to 5.9 in different preparations.
  • the green juice was divided into equal aliquots of 500 ml each. One aliquot was kept at 25° C while the other was heated to 50° C for 10 minutes. Then both aliquots were simultaneously centrifuged in a Beckmann Ultra Centrifuge in an R-21 rotor at 18,000 RPM for 30 minutes. This high centrifugal force removed all of the green color as a precipitate, leaving a clear "brown juice". Each aliquot of brown juice, i.e., the heated and unheated aliquots, was divided into equal parts, one part stored at 8° C and the other allowed to stand at 25° C. Equal amounts of Fraction 1 protein crystallized from each aliquot.
  • Example 2 Using the procedure of Example 1, a green juice having a pH of 5.7 was obtained from tobacco plants 18" to 24" in height. The green juice was divided into two equal partions. The pH of one portion was adjusted to pH
  • Example 2 Using the process of Example 1, a green juice was obtained from very young tobacco plants less than 12" tall. The juice obtained in this way had a pH of 6.0. The total juice obtained was divided into two equal parts and each part was subdivided into four equal aliquots for further processing. One aliquot from each of the orig ⁇ inal parts having a pH of 6.0 served as a control. An aliquot from each part was treated with hydrochloric acid
  • the present invention provides a convenient process for obtaining protein, and especially Fraction 1 protein, from plant material.
  • the process of the present invention obviates the need for costly and elaborate molecular filtration and Sephadex columns as required by prior art processes.
  • no chemical agent is required other than the reducing agent which, in the case of 2-mercaptoethanol, evaporates during processing or is driven off in the heating step if used, in order to obtain the Fraction 1 protein and the acid used to adjust the pH of the liquid. Because it is unnecessary to dilute the liquid, recovery of Fraction 2 proteins is also simplified.
  • the liquid portion still contains low- molecular weight compounds of value that can be more economically recovered than would be the case using the residue obtained by prior art processes since they are in their natural form and undiluted.
  • the residues obtained from prior art processes are contami ⁇ nated by the chemicals used in the process and have been diluted during separation of the Fraction 1 protein which complicates further recovery.
  • Our improved process also makes it unnecessary in most cases to use the heating regimen described in our copending application Serial No. 78,505.

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  • Life Sciences & Earth Sciences (AREA)
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PCT/US1981/000655 1981-05-18 1981-05-18 Process for isolation of proteins from plant leaves WO1982004066A1 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
BR8109020A BR8109020A (pt) 1981-05-18 1981-05-18 Processo para isolamento de proteinas a partir de folhas de plantas
DE3152847T DE3152847C2 (en) 1981-05-18 1981-05-18 Ribulose-1,5-diphosphate carboxylase and process for obtaining it
GB08300606A GB2110221B (en) 1981-05-18 1981-05-18 Process for isolation of proteins from plant leaves
PCT/US1981/000655 WO1982004066A1 (en) 1981-05-18 1981-05-18 Process for isolation of proteins from plant leaves
JP50228781A JPS58500737A (ja) 1981-05-18 1981-05-18 植物の葉から蛋白質の単離法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
WOUS81/00655810518 1981-05-18
PCT/US1981/000655 WO1982004066A1 (en) 1981-05-18 1981-05-18 Process for isolation of proteins from plant leaves

Publications (1)

Publication Number Publication Date
WO1982004066A1 true WO1982004066A1 (en) 1982-11-25

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PCT/US1981/000655 WO1982004066A1 (en) 1981-05-18 1981-05-18 Process for isolation of proteins from plant leaves

Country Status (5)

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JP (1) JPS58500737A (enrdf_load_stackoverflow)
BR (1) BR8109020A (enrdf_load_stackoverflow)
DE (1) DE3152847C2 (enrdf_load_stackoverflow)
GB (1) GB2110221B (enrdf_load_stackoverflow)
WO (1) WO1982004066A1 (enrdf_load_stackoverflow)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103734905A (zh) * 2014-01-03 2014-04-23 广东中烟工业有限责任公司 一种去蛋白烟草提取物的制备方法及应用
US11166485B2 (en) 2013-03-14 2021-11-09 R.J. Reynolds Tobacco Company Protein-enriched tobacco-derived composition
WO2021235930A1 (en) 2020-05-18 2021-11-25 Coöperatie Koninklijke Cosun U.A. Process for isolating soluble functional proteins from plant material
US11284632B2 (en) 2012-12-24 2022-03-29 Stichting Wageningen Research Economical process for the isolation of functional protein from plants
US11896030B2 (en) 2013-05-17 2024-02-13 R.J. Reynolds Tobacco Company Tobacco-derived protein compositions

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2500335B2 (ja) * 1991-11-14 1996-05-29 農林水産省農業研究センター所長 植物中または植物体表面上の微量物質の検出方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2600903A (en) * 1948-03-26 1952-06-17 Miller Harry Method of processing alfalfa
US3780183A (en) * 1971-01-25 1973-12-18 Edwards G Wheeler Plant protein product and process
US3823128A (en) * 1972-05-16 1974-07-09 Us Agriculture Preparation of edible protein from leafy green crops such as alfalfa
US4268632A (en) * 1979-09-24 1981-05-19 Leaf Proteins, Inc. Process for isolation of ribulose 1,5-diphosphate carboxylase from plant leaves

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2600903A (en) * 1948-03-26 1952-06-17 Miller Harry Method of processing alfalfa
US3780183A (en) * 1971-01-25 1973-12-18 Edwards G Wheeler Plant protein product and process
US3823128A (en) * 1972-05-16 1974-07-09 Us Agriculture Preparation of edible protein from leafy green crops such as alfalfa
US4268632A (en) * 1979-09-24 1981-05-19 Leaf Proteins, Inc. Process for isolation of ribulose 1,5-diphosphate carboxylase from plant leaves

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, volume 5, number 7, issued July 1976, Easton, Pennsylvania, (USA), PAULSEN et al.: "Spinach Ribose Diphosphate Carboxylase I. Purification and Properties of the Enzyme", pages 2350 to 2357 *
METHODS IN ENZYMOLOGY, COLOWICK et al.: Editors, volume 69, 1980, Academic Press, New York, N.Y., (USA), Section (30), "Crystallization and Assay Procedures of Tobacco Ribulose-1,5-Biophate Carboxylase-Oxygenase", by KUNG et al.: pages 326 to 336 *
SCIENCE, vol. 204, 06 April 1979, Washington, D.C., (USA) JOHAL et al.: "Crystalline Ribulose 1,5-Biphosphate Carboxylase-Oxygenase from Spinach", pages 75 to 77 *
SCIENCE, volume 176, issued 1972, Washington, D.C., (USA), CHAN et al.: "Crystalline Fraction I. Protein: Preparation in Large Yield", pages 1145-1146 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11284632B2 (en) 2012-12-24 2022-03-29 Stichting Wageningen Research Economical process for the isolation of functional protein from plants
US11166485B2 (en) 2013-03-14 2021-11-09 R.J. Reynolds Tobacco Company Protein-enriched tobacco-derived composition
US11375741B2 (en) 2013-03-14 2022-07-05 R.J. Reynolds Tobacco Company Protein-enriched tobacco-derived composition
US11896030B2 (en) 2013-05-17 2024-02-13 R.J. Reynolds Tobacco Company Tobacco-derived protein compositions
CN103734905A (zh) * 2014-01-03 2014-04-23 广东中烟工业有限责任公司 一种去蛋白烟草提取物的制备方法及应用
WO2021235930A1 (en) 2020-05-18 2021-11-25 Coöperatie Koninklijke Cosun U.A. Process for isolating soluble functional proteins from plant material
NL2025601B1 (en) 2020-05-18 2021-12-03 Cooeperatie Koninklijke Cosun U A Process for isolating soluble functional proteins from plant material

Also Published As

Publication number Publication date
DE3152847T1 (de) 1983-07-07
GB2110221A (en) 1983-06-15
DE3152847C2 (en) 1990-04-05
GB2110221B (en) 1984-10-03
BR8109020A (pt) 1983-04-12
GB8300606D0 (en) 1983-02-09
JPH03998B2 (enrdf_load_stackoverflow) 1991-01-09
JPS58500737A (ja) 1983-05-12

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