CA2236894A1 - Process to recover polyesters from transgenic plants - Google Patents

Abstract

Polyhydroxyalkanoate (PHA) polyester is recovered from vegetable matter associated with its production in transgenic plants. The process involves contacting the vegetable matter with one or more reagents inactive toward PHA
but effective to solubilize at least part of the vegetable matter, taking off the resulting solution, and recovering PHA from the residue enriched therein.
Effective solubilizing reagents that are preferred include enzyme formulations comprising carbohydrases, hydrolases or proteases.

Description

CA 02236894 1998-0~-0~
WO 97tl7459 PCT/US96/17757 PROCESS TO RECOVER POLYESTERS FRO~[ TRANSGENIC PLANTS
THIS INVENTION relates to polyester production and in particular to a microbiological process of m~king polyhydroxy~lk~no~te (PHA).
It has been recently proposed to make PHA in the metabolism of genetically modified plants, for example Arabidopsis thaliana (Poirier et al. Science 1992 Apr. 256, 520-522;
FEMS Microbiology Reviews 1992, 103, 237-246) or oil-bearing plants (GB Applications 9108756 and 9223332). The PHA may be laid down in the starchy parts of the plant or, more conveniently, in the seed, possibly in the course of germin~tion of such seed. These proposals, while identifying routes to PHA, do not specify how PHA may be recovered at a useful level of purity.
ACCORDING TO THE INVENTION PHA is recovered from vegetable matter associated with it by solubilizing said matter. More particularly, the process comprises:
(a) con1~rtin~ said matter with one or more reagents inactive toward said PHA but effective to solubilize at least part of said matter;
(b) taking offthe resllltinp solution, and (c) recovering PHA from the residue enriched therein.
In practice, for better control and better utilization of the solution(s) as by-products, the process may be carried out in successive steps, each using a different reagent, and with take-off of the solution from one such step before carrying out the next.
The solubilization steps include at least one of the following:
(i) carbohydrate solubilization;
(ii) glycopeptide solubilization;
(iii) nucleic acid solubilization; or (iv) protein solubilization.
Typically step (i) is carried out enzymatically, since chemical solubilization of carbohyd~dles such as cellulose or hemicelluloses l~uh._s vigorous con-lition~ such as the action of a solution of strong acid at 100~C or more; however, part-removal of less resistant carbohydrates such as starch may be carried out chemically. If desired, cellulose may be left undecomposed and removed later. Suitable enzymes are selected from carbohydrases.
Preferably a lllixlule of such enzymes is used, such as those exemplified below. The enzymes CA 02236894 1998-0~-0~

may be used alone or in combination with living org~ni~m~ such as bacteria or fungi of which they are constituents.
Typically step (ii) is carried out enzymatically using for example a hydrolase such as Iysozyme. Tt;~ eldL~es in the range of 15-50~C are suitable.
Typically step (iii) is carried out chemically, using an oxidant, in presence of a s~ ct~nt as described in co-pending application WO 94/10289. The temperature is typically 20-80~C. Although nucleic acids constitute only a few percent of the vegetable matter, removal of them is useful in decreasing the viscosity of the aqueous s~lcp~ncion being treated.
Step (iv) can be carried out chemically or enzymatically or a combination thereof.
~hemic~l methods include simply displacing the protein from the PHA ~nl-les by digestion with a sllrf~ct~nt, this is desirable if a substantial proportion of protein is present and/or protein is to be recovered. An ~ltern~tive chemical method involves oxidation using, for mple a peroxide or hypoehlorite Suitable tempelaL~ s for such chemical methods are in the range 50-1 00~C. If an enzymatic method is used, the enzyme is a protease. If desired, protein may be partly solubilized by protease, then more completely by oxidation.
Pl~dlol ~ to any of these steps the vegetable matter or residue may be subjected to dry- or wet- grinding or cooling, to make it more t~ec~ihle to the reagent.
Which of these is used depends on the form in which the PHA is to be recovered.
Thus, at least (i) and (iv) are used if the PHA is to be recovered as particles or latex.
However (i) suffices if the PHA is recovered in ingot form by heating the aqueous dispersion of PHA- cO~ vegetable matter to above the melting point of the PHA, or (in presence of a PHA solvent having ~ l solubility in water) to a tel~ dLu-e at which it forms a CA 02236894 1998-0~-0~

separate liquid phase, or to a t~ lalu~e at which the PHA agglomerates into easily filterable patticles.
The vegetable matter is suitably the seeds of one or more of the oil-bearing plants rape, canola, soya, sunflower, maize, oil palm and coconut, as genetically modified for example by introduction of genes encoding enzymes catalytic for the production of PHA
together with gene regulatory sequences directing ex~ sions to target plant cell components.
The required genes are preferably isolated from a PHA - acc--m~ ting procaryote such as Alcaligenes eutrophus or Chromatium vinosum. Usually such oil seeds contain oil as well as PHA, hence the process of the invention is preferably preceded by a step of recovering oil, for example by extraction into he~nP There may be a step of germination, suitably to the extent of malting, before oil extraction, for seeds that accllm~ te PHA in such a step. The above-mentioned steps of grin-ling and cooling usually precede oil extraction.
In a patticular forsn of the invention step (i) is carried out by a so-called biorefinery process, which comprises:
(a) grin-ling and cooking the seed;
(b) enzymatically solubilizing carbohydrate;
(c) allowing the resulting product to separate into the layers (from top to bottom) oil, emulsion, aqueous carbohydrate syrup, protein and hulls;
(d) ~le~smting said layers to give distinct fractions; and (e) recovering PHA from the fraction(s) in which it occuts.
The PHA produced by the process is R-stereospecific and capable of a level of cryst~llinity in the range 30-70% w/w. Its molecular weight is for example over 50,000, especially over 100,000 and up to e.g. 2 x 106.

EXAMPLES
(a) Spent rape seed meal, the residue of conventional oil extraction by cooling, crushing and extraction with hç~r~nf~, was mixed with half its weight of microbiologically r produced BIOPOLTM PHB homopolymer particles (...,u average, crystalline). The ~Lwc~ was suspended in 5 times its weight of water and adjusted to pH 7, 40~C. An enzyme solution co. .l~ g a range of carbohydrase activity (VISCOZYMETM
multienzyme complex, from Novo Nordisk, comprising ar~b~n~eç, cellulase, ,B-gl~ n~cP~ hemi-cellulose and xylanase) was added to give a 1% w/w concentration on the rape seed meal. the total mixture was stirred at pH 7, 40~C for 25h. Samples were taken at intervals and filtered. The solid retained as the filter was dried and weighed, and the weight of the dried solid was compared with the weight present in a quantity of starting mixlul~ equal to that of the sample.
(b) Run (a) was repeated using EXTRAZYMETM mllltiPn7~me complex, also ~om Novo Nordisk, co..~;.~ g ar~n~eç7 cell~ ee, ~3-gl~ n~eee, hemi-cell~ ee, pectin~ee and xylanase.
The table shows the extent to which rape seed meal was solubilized.
T~-ble Time,h 0 5 10 15 20 25 Wt Loss A % 0 40 44 48 49 50 Wt Loss B % 0 30 36 46 46 48

Claims (6)

1. A process to recover polyhydroxyalkanoate (PHA) from vegetable matter comprising:
(a) contacting said matter with one or more carbohydrase enzymes effective to solubilize at least part of said matter;
(b) taking off the resulting solution; and (c) recovering PHA from the residue enriched therein.

2. A process according to claim 1 wherein the enzymatic solubilization step further comprises/includes the use of a hydrolase or protease.

3. A process according to claim 1 wherein the solubilization reagent further comprises an oxidant.

4. A process according to claim 3 wherein the oxidant is a peroxide or hypochlorite.

5. A process to recover polyhydroxyalkanoate (PHA) from seeds comprising:
(a) grinding and cooking the seed;
(b) enzymatically solubilizing carbohydrate using a carbohydrase enzyme;
(c) allowing the resulting product to separate into the layers (from top to bottom) oil, emulsion, aqueous carbohydrate syrup, protein and hulls;
(d) decanting said layers to give distinct fractions;
and (e) recovering PHA from the fraction(s) in which it occurs.

6. A process according to claim 5 wherein enzymatic solubilization step further comprises/includes the use of a hydrolase or protease.