WO1997017459A1

WO1997017459A1 - Process to recover polyesters from transgenic plants

Info

Publication number: WO1997017459A1
Application number: PCT/US1996/017757
Authority: WO
Inventors: John Macdonald Liddell
Original assignee: Monsanto Co
Current assignee: Monsanto Co
Priority date: 1995-11-08
Filing date: 1996-11-06
Publication date: 1997-05-15
Anticipated expiration: 1998-05-08
Also published as: AU1157397A; EP0859859A1; JP2000500966A; HUP9902019A2; MX9803700A; GB9522896D0

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

PROCESS TO RECOVER POLYESTERS FROM TRANSGENIC PLANTS

THIS INVENTION relates to polyester production and in particular to a microbiological process of making polyhydroxyalkanoate (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 ofthe plant or, more conveniently, in the seed, possibly in the course of germination 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) contacting said matter with one or more reagents inactive toward said PHA but effective to solubilize at least part of said matter;

(b) taking off the resulting solution; and

(c) recovering PHA from the residue enriched therein.

In practice, for better control and better utilization ofthe 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 ofthe 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

carbohydrates such as cellulose or hemicelluloses requires vigorous conditions 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 mixture of such enzymes is used, such as those exemplified below. The enzymes may be used alone or in combination with living organisms such as bacteria or fungi of which

they are constituents.

Typically step (ii) is carried out enzymatically using for example a hydrolase such as

lysozyme. Temperatures in the range of 15-50°C are suitable.

Typically step (iii) is carried out chemically, using an oxidant, in presence of a

surfactant, as described in co-pending application WO 94/10289. The temperature is

typically 20-80°C. Although nucleic acids constitute only a few percent ofthe vegetable

matter, removal of them is useful in decreasing the viscosity ofthe aqueous suspension being

treated.

Step (iv) can be carried out chemically or enzymatically or a combination thereof.

Chemical methods include simply displacing the protein from the PHA granules by digestion

with a surfactant; this is desirable if a substantial proportion of protein is present and/or

protein is to be recovered. An alternative chemical method involves oxidation using, for

example a peroxide or hypochlorite. Suitable temperatures for such chemical methods are in

the range 50-100°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.

Preparatory to any of these steps the vegetable matter or residue may be subjected to

dry- or wet- grinding or cooling, to make it more accessible 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- containing vegetable matter to above the melting point ofthe PHA, or (in presence

of a PHA solvent having substantial solubility in water) to a temperature at which it forms a separate liquid phase, or to a temperature at which the PHA agglomerates into easily filterable

particles.

The vegetable matter is suitably the seeds of one or more ofthe 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 expressions to target plant cell components.

The required genes are preferably isolated from a PHA - accumulating procaryote such as

Alcaligenes eutrophus or Chromatium vinosum. Usually such oil seeds contain oil as well as

PHA, hence the process ofthe invention is preferably preceded by a step of recovering oil, for

example by extraction into hexane. There may be a step of germination, suitably to the extent

of malting, before oil extraction, for seeds that accumulate PHA in such a step. The above-

mentioned steps of grinding and cooling usually precede oil extraction.

In a particular form ofthe invention step (i) is carried out by a so-called biorefinery

process, which comprises:

(a) grinding 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) decanting said layers to give distinct fractions; and

(e) recovering PHA from the fraction(s) in which it occurs.

The PHA produced by the process is R-stereospecific and capable of a level of

crystallinity 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 IO⁶. EXAMPLES

(a) Spent rape seed meal, the residue of conventional oil extraction by cooling, crushing

and extraction with hexane, was mixed with half its weight of microbiologically

produced BIOPOL™ PHB homopolymer particles (...μ average, crystalline). The

mixture was suspended in 5 times its weight of water and adjusted to pH 7, 40°C. An

enzyme solution containing a range of carbohydrase activity (VISCOZYME™

multienzyme complex, from Novo Nordisk, comprising arabanase, cellulase, β-

glucanase, 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 ofthe dried solid was compared with the weight present in a quantity of starting mixture equal to that ofthe sample.

(b) Run (a) was repeated using EXTRAZYME™ multienzyme complex, also from Novo

Nordisk, containing arabanase, cellulase, β-glucanases, hemi-cellulase, pectinase and

xylanase.

The table shows the extent to which rape seed meal was solubilized.

Table

Time, h 0 5 10 15 20 25 1

Wt Loss A % 0 40 44 48 49 50

Wt Loss B % 0 30 36 46 46 48 |

Claims

WHAT IS CLAIMED IS:

1. A process to recover polyhydroxyalkanoate (PHA) from vegetable matter comprising:

(a) contacting said matter with one or more reagents inactive toward said PHA but

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. The process of claim 1 wherein the solubilization reagent comprises an enzyme.

3. The process of claim 2 wherein the enzymatic solubilization reagent comprises a

carbohydrase, hydrolase or protease.

4. The process of claim 1 wherein the solubilization reagent comprises an oxidant.

5. The process of claim 4 wherein the oxidant is a peroxide or hypochlorite.

6. A process to recover polyhydroxyalkanoate (PHA) from seeds comprising:

(a) grinding 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) decanting said layers to give distinct fractions; and

(e) recovering PHA from the fraction(s) in which it occurs.

7. The process of claim 6 wherein enzymatic solubilization step includes a carbohydrase,

hydrolase or protease solubilization reagent.

8. A polyhydroxyalkanoate (PHA) recovered by the process of claims 1 -6, which PHA is

R-stereospecific and capable of a level of crystallinity in the range of 30 to 70% w/w, and has

a molecular weight greater than 50,000 and up to 2 x IO⁶.