Methods for recovery of leaf proteins

Patent metadata

Field	Value
Publication number	US-9321806-B2
Application number	US-45142908-A
Country	US
Kind code	B2
Filing date	May 15, 2008
Priority date	May 16, 2007
Publication date	Apr 26, 2016
Grant date	Apr 26, 2016

Abstract

A novel method for processing soluble plant leaf proteins is described. While leaf proteins are considered potentially the most abundant source of protein in nature, the lack of efficient processing techniques for leaf proteins has limited their commercial use. The method described in this patent provides a means of extracting and purifying leaf proteins from plants which is suitable for leaf protein production on an industrial scale.

First claim

What is claimed is:

1. A method for simultaneously recovering both ribulose-1,5-bisphosphate (RUBP) carboxylase/oxygenase (“rubisco”) and non-rubisco soluble leaf proteins from plant leaves, which method is conducted throughout at a temperature between about 0°-about 25° Celsius, comprising the steps of:
(a) simultaneously disrupting the cell walls of plant leaves and contacting rubisco and non-rubisco soluble proteins released from the disrupted plant leaves with a buffer solution, so that disrupted leaf materials and rubisco and non-rubisco soluble leaf proteins are exposed to the buffer solution and both the rubisco and non-rubisco soluble leaf proteins solubilize and are kept in the buffer solution,
wherein the buffer solution (i) has a solute concentration between 0.025 M and 0.3 M; (ii) is present in a buffer-to-leaf ratio of greater than approximately 1:2 but not more than approximately 8:1; (iii) has a pH between 6.5 and 9.0; (iv) has a buffering region that is effective within the pH value of (iii); and (v) optionally comprises a chelating agent and/or a reducing agent;
(b) removing from the buffer solution produced in step (a) substantially all cellulosic material to produce a buffer solution containing plant chloroplast material and the rubisco and non-rubisco soluble leaf proteins, which proteins remain solubilized therein,
which step (b) occurs without adsorption on a solid support or removal by filtration of the rubisco or non-rubisco soluble leaf proteins;
(c) removing from the buffer solution produced in step (b) at least 80% of the plant chloroplast material to produce a buffer solution containing the rubisco and non-rubisco soluble leaf proteins, which proteins remain solubilized therein,
which step (c) occurs without adsorption on a solid support or removal by filtration of the rubisco or non-rubisco soluble leaf proteins;
wherein throughout steps (a), (b) and (c) both the rubisco and non-rubisco soluble leaf proteins remain solubilized and kept in the buffer solution while cellulosic material and plant chloroplast material are sequentially removed therefrom, and
(d) drying down together in one product both the solubilized rubisco and non-rubisco soluble leaf proteins from the buffer solution, wherein said method further comprises steps between step (c) and step (d):
(aa) precipitating without denaturing soluble leaf proteins by conducting an isoelectric point precipitation on the buffer solution containing the solubilized leaf proteins, for up to 40 minutes at a pH of 5.3 equivalent to the isoelectric point of the soluble leaf proteins, or within .5 pH units of pH 5.3 of the isoelectric point;
(bb) removing any supernatant; and
(cc) resuspending the precipitated soluble leaf proteins in the buffer solution.

2. The method of claim 1 , wherein in step (a) the cell walls of the leaves are disrupted by chopping, milling, grinding or crushing the leaves, pulping, maceration procedures, mechanical pressure, rollers or homogenizing.

3. The method of claim 1 , wherein after step (a) the soluble leaf proteins are maintained in the buffer solution for up to twenty-four hours.

4. The method of claim 3 , wherein the soluble leaf proteins are maintained in the buffer solution at ambient temperature or lower.

5. The method of claim 1 , wherein the buffer solution is a system suitable for protein extraction, selected from the group consisting of the combinations: sodium phosphate dibasic and potassium phosphate monobasic (Na 2 HPO 4 —KH 2 PO 4 ), potassium phosphate monobasic/sodium hydroxide, sodium hydroxide/citric acid, acetic acid/ammonium acetate, potassium hydroxide/potassium phosphate monobasic, citric acid/disodium phosphate, potassium phosphate monobasic/potassium phosphate, dibasic, potassium acid phthalate/sodium hydroxide, potassium carbonate/potassium tetraborate/potassium hydroxide/disodium EDTA dihydrate, giordano's buffer, sodium acetate trihydrate/sodium chloride, tris(hydroxymethyl)aminomethane (Tris), EDTA/Tris/HC 1 , 2-amino-2-(hydroxymethyl)-1,3-propanediol/Tris, Tris/EDTA, ammonium chloride/ammonium hydroxide, HEPES/NaC 1 , imidazole, phosphate, N-morpholinopropane sulfonic acid (MOPS), N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (“TES”), triethanolamine, and N-tris(hydroxymethyl)-methyl-glycine (“Tricine”).

6. The method of claim 1 , wherein the chelating agent is selected from the group consisting of EDTA, EGTA, HEDTA, DTPA, NTA, calcium citrate, calcium diacetate, calcium hexametaphosphate, citric acid, gluconic acid, dipotassium phosphate, disodium phosphate, isopropyl citrate, monobasic calcium phosphate, monoisopropyl citrate, potassium citrate, sodium acid phosphate, sodium citrate, sodium gluconate, sodium hexametaphosphate, sodium metaphosphate, sodium phosphate, sodium pyrophosphate, sodium tripolyphosphate, stearyl citrate, tetra sodium pyrophosphate, calcium disodium ethylene diamine tetra-acetate, glucono delta-lactone, potassium gluconate and the like, and their analogs, homologs and derivatives.

7. The method of claim 1 , wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, 2-mercaptoethylamine-HCL, cysteine-HCL, Ellman's reagent, BME, DTT, glutathione, cystein, Tris (2-carboxyethyl) phosphine hydrochloride, TCEP disulfide, n-ethylmaleimide, TCEP-HCL, ferrous ion, lithium aluminum hydride (LiAlH 4 ), nascent hydrogen, sodium amalgam, sodium borohydride (NaBH 4 ), stannous ion, sulfite compounds, hydrazine, zinc-mercury amalgam (Zn(Hg)), diisobutylaluminum hydride, lindlar catalyst, oxalic acid (C 2 H 2 O 4 ), dithioerythritol, thioglycolate, glutathione, cysteinem ascorbate, and their analogs, homologs and derivatives.

8. The method of claim 1 , wherein in step (b) the cellulosic material is removed using a screw press, fly presser, blender, mechanical presser, mechanical dewatering device, or crusher.

9. The method of claim 1 , wherein in step (c) the chloroplast material is removed using a centrifuge.

10. The method of claim 1 , wherein in step (d) the drying down is done by spray drying, vacuum drying, or freeze drying.

11. The method of claim 1 , wherein the plant leaves are from tobacco plants, plants of the species Nicotiana , alfalfa, ryegrass, tomato, spinach or potato.

12. The method of claim 11 , wherein the plant leaves are from tobacco plants.

13. The method of claim 12 , wherein the buffer solution comprises a buffer system of Na 2 HPO 4 and KH 2 PO 4 at a concentration of approximately 0.067 M, 10 mM EDTA, and 25 mM 2-mercaptoethanol, the buffer-to-leaf ratio is between 3:1 and 8:1, and the temperature is between approximately 4° and approximately 10° Celsius.

14. The method of claim 1 , wherein the plant leaves are from tobacco plants, which comprises the further steps between step (c) and step (d):
precipitating soluble leaf proteins by conducting an isoelectric point precipitation on the buffer solution containing the solubilized leaf proteins, for up to 40 minutes at a pH between about 3.7 and about 4.7;
removing any supernatant; and
resuspending the precipitated soluble proteins in the buffer solution.

15. The method of claim 1 , wherein the plant leaves are from tobacco plants, which further comprises the steps between step (c) and step (d):
conducting isoelectric point precipitation for up to 40 minutes at a pH between about 3.7 and about 4.7 on buffer solution containing the solubilized leaf proteins to precipitate soluble proteins;
collecting supernatant;
conducting a second isoelectric point precipitation for up to 40 minutes at pH of about 4.2 on the supernatant to precipitate soluble proteins pres

Assignees

• Lo Martin
• Fu Hong
• Univ Maryland

Inventors

• Lo Martin
• Fu Hong

Classifications

• A61K36/81

  Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed · CPC title

• C07K1/30

  by precipitation · CPC title

• C07K1/145

  by extraction or solubilisation · CPC title

• C07K1/36 Primary

  by a combination of two or more processes of different types · CPC title

• C12Y401/01039

  Ribulose-bisphosphate carboxylase (4.1.1.39) · CPC title