WO1997015681B1

WO1997015681B1 - Methods for isolating polyhydroxyalkanoates from plants

Info

Publication number: WO1997015681B1
Application number: PCT/US1996/016921
Authority: WO
Filing date: 1996-10-23
Publication date: 1997-06-19
Anticipated expiration: 1998-04-26

Abstract

Methods are provided for separating polyhydroxyalkanoates ('PHAs') from plants, such as transgenic oil crop plants. The methods advantageously permit both the oil and the PHAs to be recovered from the plant biomass. To isolate the PHAs, in one embodiment, a biomass derived from an oil crop plant is pre-processed, for example by grinding, crushing or rolling. The oil then is extracted from the biomass with a first solvent in which the oil is soluble and in which the PHAs are not highly soluble to remove the oil. The biomass then can be extracted with a second solvent in which the PHA is soluble, to separate the PHA from the biomass. Alternatively, the PHA-containing biomass is treated with a chemical or biochemical agent, such as an enzyme, to chemically transform the PHA into a PHA derivative. The PHA derivative then is separated from the mixture using, for example, a physical separation process such as distillation, extraction or chromatography. Advantageously, using the method, the plant oils, the PHAs and PHA derivatives can be recovered and purified on a large scale from oil containing plants such as transgenic oil crop plants.

Claims

AMENDED CLAIMS[received by the Intemationai Bureau on 9 May 1997 (09.05.97); original claims 1-37 replaced by amended claims 1-34 (7 pages)]

1. A method for separating a polyhydroxy alkanoate ("PHA") from a plant biomass comprising plant oil and meal, the method comprising extracting the plant biomass with a first solvent to essentially remove the oil from the biomass, and then separating the PHA from the plant biomass.

2. The method of claim 1 wherein the plant biomass is derived from a transgenic plant.

3. The method of claim 1 further comprising derivatizing the PHA prior to separating the PHA from the biomass.

4. The method of claim 1 wherein the separated PHA comprises one or more units having the formula:

-OCR'R^CR^CO- wherein n is 0 or an integer; and wherein R¹, R², R\ and R⁴ each are independently selected from the group consisting of hydrocarbon-radicals, halo- and hydroxy-substituted radicals, hydroxy radicals, halogen radicals, nitrogen-substituted radicals, oxygen-substituted radicals and hydrogen atoms.

5. The method of claim 4 wherein the separated PHA is selected from the group consisting of monomers, dimers, linear and cyclic oligomers, and lactones of the units.

6. The method of claim 3 wherein the separated PHA is selected from the group consisting of esters defined by the formula: HOCR'R²(CR³R⁴)_nCO₂R⁵; amides defined by the formula: HOCR¹R²(CR³R⁴)_nCONR⁵R⁶; thioesters defined by the formula: HOCR¹R²(CR³R⁴)_nCOSR⁵; acids defined by the formula: HOCR¹R²(CR³R⁴)_nCO₂H; ethers defined by the formula: R⁶OCR¹R²(CR³R⁴)_nCO₂R⁵; esters defined by the formula: R⁶CO₂CR¹R²(CR³R⁴)_nCO₂R⁵; unsaturated compounds, defined by the formulas: - 30 -

R 'R²C=CR³(CR⁴R⁵)_nCO₂R⁶; and

R'R^CCCR⁴) =C(R⁵)CO₂R⁶; diols defmed by the formula: HOCR¹R²(CR³R⁴)_nCH₂OH; lactones or macrolides defined by the formula:

H -OCR^lR²(CR³R⁴)„CO-K , wherein x is an integer; and ketones or aldehydes defined by the formulas:

HOCR'R²(CR³R⁴)_nCOR⁵;

HOCR¹R²(CR³R⁴)_nCHO;

R'CO(CR²R³)_nCOOR⁴; and

R¹CO(CR²R³)_nCOR⁴; wherein n is 0 or an integer; and wherein R^l, R², R³, R⁴, R⁵ and R⁶ are each independently selected from the group consisting of hydrocarbon radicals, halo- and hydroxy-substituted radicals, hydroxy radicals, halogen radicals, nitrogen-substituted radicals, oxygen-substituted radicals, and hydrogen atoms.

7. The method of claim 1 comprising a) providing a plant biomass containing a PHA; b) pre-processing the plant biomass to obtain a mixture containing PHA, oil and plant meal; c) extracting oil from the mixture with a first solvent at a temperature at which the oil is soluble and in which the PHA is not highly soluble, to obtain a residual meal mixture comprising PHA; and d) extracting the residual meal mixture obtained in step c) with a second solvent in which the PHA is soluble, to separate PHA from the biomass.

8. The method of claim 1 comprising: a) providing a plant biomass containing a PHA; b) pre-processing the plant biomass to produce a mixture containing PHA, oil and plant meal; c) extracting oil from the mixture with a first solvent at a temperature at which the oil is soluble and in which the PHA is not highly soluble, to obtain a residual meal mixture comprising PHA; d) treating the residual meal mixture comprising PHA, obtained in step c), with at least one chemical or biochemical agent, to chemically derivatize the PHA; and e) separating derivatized PHA from the residual meal mixture obtained in step d).

9. The method of claim 7 or 8 wherein step b) comprises pre¬ processing the plant biomass using one or more processes selected from the group consisting of drying, dehulling, cleaning, ageing, cleaning, weighing, cracking, flaking, pressing, rolling, grinding, cooking, crushing, settling, filtering, washing, centrifugal fractionation, and air classification.

10. The method of claim 7 wherein the second solvent is selected from the group consisting of a chlorinated organic solvent, an alkyl carbonate, an alcohol, a hydroxyacid, acetic acid, and mixtures thereof.

11. The method of claim 10 wherein the second solvent comprises a chloronated organic solvent selected from the group consisting of chloroform, methylene chloride, dichloroethane, trichloroethane, tetrachloroethane and dichloroacetate. - 32 -

12. The method of claim 7 wherein the second solvent comprises a composition selected from the group consisting of a propylene carbonate, ethylene carbonate, trifluoroethanol, acetic anhydride, acetic acid, dimethylformamide, ethylacetoacetate, triolein, toluene, dioxane, tetrahydrofuran, diethylether, pyridine, and alcohols having more than three carbon atoms.

13. The method of claim 8 wherein, in step d), the residual meal mixture comprising PHA is treated with at least one chemical agent selected from the group consisting of acids, bases, detergents, oxidizing agents, chelating agents, reducing agents, nucleophilic reagents, electrophilic reagents, metal ions, and free radicals.

14. The method of claim 8 wherein the PHA is derivatized by a chemical transformation selected from the group consisting of esterification, transesterification, hydrolysis, saponif ication, aminolysis, thiolysis, etherification, silylation, addition, elimination, rearrangement, reduction, and condensation.

15. The method of claim 8 wherein the biochemical agent is an enzyme.

16. The method of claim 15 wherein the enzyme is selected from the group consisting of a depolymerase, protease, nuclease, lipase, phosphorylase and a glycosidase.

17. The method of claim 8 wherein, in step e), the derivatized PHA is separated by a physical process selected from the group consisting of distillation, extraction, centrifugation, filtration, evaporation, and chromatography.

18. The method of claim 7 or 8 wherein the first and second solvents each have boiling points between 30 'C and 250' C.

19. The method of claim 7 or 8 further comprising precipitating the separated PHA derivative. 33 -

20. The method of claim 1 wherein the biomass is derived from a plant source selected from the group consisting of soybean, cotton, coconuts, groundnuts, rapeseed, sunflower seed, olive, palm, sesame seed, linseed, castor, safflower seed, tobacco, corn, mustard, and potato.

21. The method of claim 4 wherein the separated PHA includes one or more of the same or different units selected from the group consisting of hydroxy butyrate, hydroxyvalerate, hydroxy hexanoate, hydroxy heptanoate, hydroxy octanoate, hydroxynonanoate, and hydroxy decanoate .

22. The method of claim 8 wherein the separated PHA is selected from the group consisting of 3-hydroxybutyric acid, 4- hydroxybutyric acid, crotonic acid and alkyl esters thereof.

23. The method of claim 2 wherein the plant biomass is derived from a plant containing a heterologous PHA synthase gene derived from a microorganism selected from the group consisting of Acinetobacter, Aeromonas, Alcaligenes, Azotobacter, Bacillus, Brevibacteriwn, Corynebacterium, Chromatium, Flavobacterium, Halobacterium, Pseudomonads, Nocardia, Rhodococcus, Thiocystis, Streptomyces, Streptococcus and Zoogloea.

24. A method for separating a polyhydroxyalkanoate ("PHA") from a plant biomass comprising plant oil, the method comprising extracting a plant biomass with a first solvent at a temperature at which the oil and PHA are soluble, to essentially remove oil and PHA from the biomass, and then separating PHA from the oil.

25. The method of claim 24 wherein the plant biomass is derived from a transgenic oil crop plant.

26. The method of claim 25 wherein the method comprises a) providing a plant biomass containing a PHA; b) pre-processing the plant biomass to obtain a mixture of PHA, oil and plant meal; - 34 -

c) extracting oil and PHA from the mixture with a first solvent in which the oil and the PHA are soluble and in which the meal is not highly soluble; and d) separating the PHA from the oil.

27. The method of claim 26 wherein the PHA is separated from the oil in step d) by treating the PHA-oil mixture with a chemical or biochemical agent, thereby to chemically derivatize the PHA, and separating derivatized PHA from the oil.

28. The method of claim 26 wherein the solvent is selected from the group consisting of a chlorinated organic solvent, an alkylcarbonate, an alcohol, a hydroxyacid and a hydrocarbon, and mixtures thereof.

29. The method of claim 26 wherein the solvent is selected from the group consisting of hexane, trifluoroethanol, acetic anhydride, dimethylformamide, ethylacetoacetate, triolein, acetic acid, toluene, dioxane, tetrahydrofuran, diethylether and pyridine.

30. The method of claim 27 wherein the biochemical agent is an enzyme selected from the group consisting of PHA depolymerases, proteases, nucleases, lipases, hydratases, phosphorylases, cellulases and glycosidases.

31. The method of claim 7, 8, 26 or 27 wherein the separated PHA includes a functional group selected from the group consisting of amides, thioesters, acids, ethers, esters, unsaturated compounds, diols, ketones and aldehydes.

32. The method of claim 7, 8, 26 or 27 wherein the separated PHA includes one or more units selected from the group consisting of a 3-hydroxyacid, a 4-hydroxyacid and a 5-hydroxyacid.

33. The method of claim 7 or 26 wherein the separated PHA is separated in an organic solvent; and wherein the method further comprises: - 35

emulsifying the PHA in the organic solvent in an aqueous solution containing a surfactant, thereby to form a PHA latex.

34. The method of claim 7 or 26 wherein the separated PHA is separated in an organic solvent and wherein the method further comprises emulsifying the PHA in the organic solvent in an aqueous solution and removing the solvent thereby forming a PHA latex.