Novel methods for recovery of leaf proteins

Patent metadata

Field	Value
Publication number	US-2016208236-A1
Application number	US-201615080669-A
Country	US
Kind code	A1
Filing date	Mar 25, 2016
Priority date	May 16, 2007
Publication date	Jul 21, 2016
Grant date	—

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

1 - 16 . (canceled)

17 . A method for recovering ribulose 1.5-bisphosphate carboxylase crystals from tobacco plant leaves, at a temperature between about 0°-about 25° Celsius, comprising the steps of:
(a) in the presence of a buffer solution, disrupting the cell walls of plant leaves containing soluble proteins so that soluble leaf protein is exposed to the buffer solution and solubilizes therein, wherein the buffer solution has a pH between about 6.5 and about 8.9, and is present in a buffer-to-leaf ratio of greater than approximately 1:2, and comprises a buffer system 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/Hcl, 2-amino-2-(hydroxymethyl)-1,3-propanediol/Tris, Tris/EDTA, ammonium chloride/ammonium hydroxide, HEPES/NaCl, imidazole, phosphate, N-morpholinopropane sulfonic acid (MOPS), N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (“TES”), triethanolamine, and N-tris(hydroxymethyl)-methyl-glycine (“Tricine”)
optionally, a chelating agent, and
optionally, a reducing agent,
(b) removing substantially all cellulosic material while the soluble leaf proteins remain solubilized in the buffer solution;
(c) removing the majority of plant chloroplast material while the soluble leaf proteins remain solubilized in the buffer solution,
(d) conducting isoelectric point precipitation at a pH between about 3.7 and about 4.7 on buffer solution containing the solubilized leaf proteins to precipitate soluble proteins, where a supernatant containing a clear portion is produced,
(e) collecting the clear portion of the supernatant,
(f) adding ammonium sulfate to the clear portion of the supernatant,
(g) precipitating the solution of (f) and removing the precipitated material,
(h) adding ammonium sulfate to the solution remaining of (g),
(i) allowing the solution of (h) to precipitate and collecting the precipitated material,
(j) resuspending the precipitated material of (i) in the buffer solution and allowing the product to remain in resuspension, and
(k) crystallizing and collecting ribulose 1.5-bisphosphate carboxylase from the solution of (j), wherein the resultant crystals are at least 90% pure.

18 . A method for sequentially removing cellulosic material and plant chloroplast material from plant leaf material without also removing ribulose-1,5-bisphosphate (RUBP) carboxylase/oxygenase (“rubisco”) and non-rubisco soluble leaf proteins,
which method is conducted throughout at a temperature between about 0°-about 25° Celsius,
comprising the steps of:
(a) disrupting the cell walls of plant leaves in the presence of and in contact with buffer solution, under conditions that cellulosic material, plant chloroplast material, rubisco and non-rubisco soluble proteins are released into the buffer solution,
wherein the rubisco and non-rubisco soluble proteins are solubilized in the buffer solution, and
wherein the buffer solution (i) has a solute concentration between 0.025M and 0.3M; (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) at least 99% of the cellulosic material,
(c) removing from the buffer solution produced in step (b) at least 95% of the plant chloroplast material so as to produce a buffer solution substantially free of cellulosic material and plant chloroplast material, but containing the rubisco and non-rubisco soluble leaf proteins, which proteins remain solubilized therein,
wherein throughout all of steps (a), (b) and (c) both the rubisco and non-rubisco soluble leaf proteins are in a solubilized state within the buffer solution, and are not intentionally removed therefrom,
and
(d) drying down together in one product both the solubilized rubisco and non-rubisco soluble leaf proteins from the buffer solution.

19 . The method of claim 18 , wherein in step (d) the product is made up of at least 95% rubisco and non-rubisco soluble leaf proteins.

20 . The method of claim 18 , wherein in step (d) the product is made up of at least 99% rubisco and non-rubisco soluble leaf proteins.

21 . The method of claim 18 , wherein after step (a) the cellulosic material, plant chloroplast material, rubisco and non-rubisco soluble proteins are maintained in the buffer solution for up to twenty-four hours.

22 . The method of claim 18 , 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/HCl, 2-amino-2-(hydroxymethyl)-1,3-propanediol/Tris, Tris/EDTA, ammonium chloride/ammonium hydroxide, HEPES/NaCl, imidazole, phosphate, N-morpholinopropane sulfonic acid (MOPS), N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (“TES”), triethanolamine, and N-tris(hydroxymethyl)-methyl-glycine (“Tricine”).

23 . The method of claim 18 , wherein in step (d) the drying down is done by spray drying, vacuum drying, or freeze drying.

24 . The method of claim 18 , which comprises the further steps between step (c) and step (d):
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, or within 0.5 pH units of pH 5.3; removing any supernatant; and resuspending the precipitated soluble leaf proteins in the buffer solution.

25 . The method of claim 18 , wherein the plant leaves are from tobacco plants, plants of the species Nicotiana , alfalfa, ryegrass, tomato, spinach or potato.

26 . The method of claim 18 , wherein the plant leaves are from tobacco plants, and wherein the buffer solution comprises a buffer system of Na 2 HPO 4 and KH 2 PO 4 at a concentration of approximately 0.067M, 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.

27 . The method of claim 18 , 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.

Assignees

• Univ Maryland

Inventors

• Lo Martin
• Fu Hong

Classifications

• B01D11/0288

  Applications, solvents · CPC title

• B01D9/0059

  General arrangements of crystallisation plant, e.g. flow sheets · CPC title

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12Y401/01039

  Ribulose-bisphosphate carboxylase (4.1.1.39) · CPC title

• C07K1/145

  by extraction or solubilisation · CPC title