WO2022195035A2 - Process of obtaining a purified rubisco preparation from a photosynthetic material - Google Patents

Abstract

A process of obtaining a RuBisCO preparation from photosynthetic material, wherein the process comprises the steps of: - lysing said photosynthetic material to extract RuBisCO accompanied by a fraction of chlorophylls of said photosynthetic material;- performing precipitation with sodium phytate and/or calcium chloride; - obtaining a phytate-calcium-RuBisCO insoluble ternary complex; and - performing solubilization of said complex.

Description

Process of obtaining a purified RuBisCO preparation from a photosynthetic material

Field of the invention

Aspects of the invention relate to the process for obtaining a RuBisCO preparation from a photosynthetic material.

RuBisCO is to be interpreted broadly and may include at least the following definitions: ribulose-bisphosphate carboxylase, RuBP carboxylase, diphosphoribulose carboxylase, ribulose 1,5 -diphosphate carboxylase, carboxydismutase, ribulose 1,5 -biphosphate carboxylase and ribulose l,5-di(or bis) phosphate carboxylase-oxygenase, 3-phospho-D- glycerate carboxy-lyase (dimerizing; D-ribulose 1,5-bisphosphate-forming); 3-phospho- D-glycerate carboxy-lyase (dimerizing).

Preferred aspects concern the large scale purification of a protein for human and animal consumption.

Background to the invention

The worldwide need for sustainable protein production for human and animal feed has never been greater. In addition, there is also an increasing demand for plant derived protein. Many attempts have focused on developing a large-scale strategy capable of purifying at a reasonable price the most abundant protein in nature, RuBisCO (E.C. 4.1.1.39): the enzyme catalyzing the first reactions of two metabolically opposed pathways, the Calvin cycle and photorespiration. Each molecule of plant RuBisCO is a hexadecamer composed of eight large subunits (LSU; ca. 475 amino acid residues or 50 to 55 kDa each) and eight small subunits (SSU; ca. 125 amino acid residues or 12 to 15 kDa each). It is therefore a very large protein, composed of approximately 4,800 amino acid residues and a molecular mass of about 550 kDa. RuBisCO is recognized as a sluggish catalyst with a turnover number around 15 CO2 molecules fixed per s per enzyme molecule, a number which drops to ca. 3 under optimum agricultural conditions - compare to catalase, where a single enzyme molecule can decompose many millions of hydrogen peroxide molecules per second under optimal conditions. To cope with the unavoidable extremely low RuBisCO catalytic efficiency and with the fact that the enzyme spends, for the vast majority of crops and depending on the environmental conditions, between 25 and virtually 100% of the time fixing oxygen in photorespiration rather than carbon dioxide in the Calvin cycle, plants accumulate massive amounts of RuBisCO in their photosynthetic tissues in order to grow at reasonable rates. For the vast majority of crops, this means that a single protein (i.e., RuBisCO) comprises typically over 50% of the total leaf proteins, where we may find over 10,000 other different proteins. In other words, RuBisCO comprises ca. 50% of the total leaf protein in almost all the plants consumed, meaning that it is ca. 50% pure in the leafy vegetables that are eaten.

Immediate consequences derived from the above comments are that everyone eating photosynthetic tissues ingests large amounts of RuBisCO. The nutritional quality of RuBisCO as an edible protein is unsurmountable:

- It is a natural compound, present in very high amounts in all green plant tissues consumed;

- It is non-toxic when ingested in any amount;

- It is readily digested by the proteases of our digestive system;

- It is non-allergenic; indeed, it is often used as a negative control in allergenicity tests;

- From the human nutritional point of view, it is the commercially available protein closer to the ideal protein, as defined by FAO (Food and Agriculture Organization of the United Nations) (chemical index = 100 when compared to the ideal protein recommended by FAO/WHO, 2007); thus, among animal proteins, egg protein is far too rich in what the branched-chain amino acids are concerned (eggs are also a source of allergens), whereas some whey proteins are potent allergens and expensive when commercialized in the pure form; among plant proteins, legume seed proteins (e.g. soybean, pea, etc.) are also potent allergens and rather poor in the sulfur-containing amino acids (cysteine and methionine), whereas cereal proteins are deficient in lysine. RuBisCO is a well-balanced protein, with a proportion of essential amino acids that equals or exceeds that recommended by FAO. Each RuBisCO molecule contains almost 200 sulfur atoms, ca. 100 derived from cysteine and ca. 100 from methionine residues.

It is believed that RuBisCO large-scale extraction and purification for further application in the food industry is a process that could solve several global problems such as protein malnutrition, a well-known public health problem (Miiller & Krawinkel, 2005; Stefano et al. , 2018). Virtually all proteins which are commercially available for human and animal consumption are integral members of the lists of the major allergens both in Europe and the US - but not RuBisCO. Based on all its well-studied characteristics, it seems clearly established that RuBisCO may be regarded as the ideal protein for both human and animal consumption. It is abundantly present in all photosynthetic tissues and is often used as a negative control in allergenicity studies. With all the information provided above, it is somewhat strange that up to this moment no one has yet developed a methodology for the non-expensive, large scale RuBisCO purification in the form of a colourless, odourless and tasteless soluble purified powder, yielding RuBisCO that may be considered, simultaneously, as organic, generally regarded as safe and obtained by a clean extraction procedure. Many procedures have been described in the literature, but they are either non- scalable due to the complexity of the purification protocol or to the huge price of the resulting pure product, or produce impure, insoluble, unpleasantly tasting and/or green preparations of RuBisCO.

Aspects of the invention provide a sustainable and scalable process for the purification of RuBisCO from photosynthetic cells/tissues/organs/entire organisms. In certain embodiments, the final product, RuBisCO, exhibits a high degree of purity and yield, and consists of a white powder, devoid of pigments (e.g., chlorophylls and carotenoids), obtained without the use of organic solvents or any other toxic compound, with no odour, smell or taste, something which has not reached the market yet. Prior art known to the applicants

Existing methods for large scale purification of RuBisCO

There are several methods of purifying RuBisCO in the laboratory, which are not amenable to adaptation for large scale purification at an industrial level and a reasonable price. This explains why, to date, no pure RuBisCO is on the market, with a multitude of potential applications. However, although there are some methods in published patent databases that apparently allow for large-scale purifications, in fact they only extract the total soluble protein or produce highly impure or insoluble and/or green-coloured RuBisCO, and there is still no simple and economical method to isolate large amounts of the pure enzyme (in the order of kg and more) based on a methodology that is compatible with the food industry.

In 1981, a crystallization method with 2-mercaptoethanol was described in US4268632, in which a purification of 90% of RuBisCO was obtained. This method requires temperature control, since protein extraction is carried out by heating to 50 °C (non-denaturing temperature for RuBisCO), and subsequent cooling to temperatures lower than room temperature, to allow adequate formation of protein crystals.

In 1982, another large-scale purification method was published in US4400471, through crystallization with PEG (polyethylene glycol). This method uses different amounts of PEG, between 8 and 13% (w/v), under controlled pH and temperature, thus allowing a purification equivalent to the method described in the previous paragraph. In the same patent publication, it is also mentioned the use of an ion exchange resin, which allows a considerable degree of purification. The author states that RuBisCO is the only compound that can bind to this resin, so its use allows a good degree of purification, after dissociation of the resin-RuBisCO complex using a solution of divalent metal ions. Alternatively, the two methods can also be combined, first crystallizing and then passing RuBisCO through the resin, thus achieving an even higher degree of purification (>90%).

The purification methods most often used to obtain RuBisCO for rheological studies involve precipitation at pH 3.5 of the soluble extract of alfalfa ( Medicago sativa ), in which a precipitation of almost all proteins is obtained by denaturation, the majority of which is RuBisCO, precipitation with ammonium sulfate and an affinity separation of spinach leaf soluble extract (WO2011078671).

Tenorio et al. in 2017 used sugar beet for the purification of proteins from their leaves, considered as a waste byproduct. The proteins were extracted by applying a precipitation at high temperatures (50 °C). The heating of proteins can cause their denaturation and should therefore be a step to be avoided. As RuBisCO is found in leaves in large quantities, it is reported that this methodology is capable of purifying RuBisCO up to 90% of the total protein - This may be an unexpected result, as RuBisCO has been claimed not to denature at 50 °C. On the other hand, the final product is expected to comprise the total soluble leaf proteins existing in the beet leaf, probably enriched in those proteins which tend to precipitate at 50 °C.

In summary, the methods that have been developed for RuBisCO purification show several disadvantages which make them inappropriate for the large scale purification required for the commercial availability of pure RuBisCO, such as: the purification procedures, which usually comprise very extensive protocols, with a high number of steps and most often unsuitable to undergo scaling-up; use of organic solvents that may cause denaturation/aggregation, decrease the solubility of RuBisCO and leave residues harmful to human health, leading to a final product that cannot be considered as (i) organic, (ii) generally regarded as safe and (iii) obtained by a clean extraction procedure (using exclusively water and ethanol as solvents); and the presence of chlorophylls, which gives a green colour to the final product.

In recent years, some companies have claimed their success in isolating this protein, although it has been found that they have only obtained a protein concentrate, where RuBisCO is the major protein. These extracts may contain compounds that will contribute negatively to their quality, namely the presence of potentially allergenic proteins, phenolic acids and tannins, as well as residues from the purification procedure itself, which may be harmful to human health.

RuBisCO-producing companies

There are currently companies that claim to purify RuBisCO on a large scale, namely France Luzerne, TNO and NIZO. However, the first obtains only a total soluble extract with a low degree of RuBisCO purification. It is worth remembering that RuBisCO is already ca. 50% pure (relative to the total leaf protein) in the leaves of C3 plants. This extract may contain potentially allergenic and/or other undesirable proteins, tannins and other phenolic compounds, which can negatively contribute to the nutritional quality of the protein concentrate.

The first company that produced RuBisCO on a large scale was France Luzerne, a French company participating in Fralupro, a European project whose objective was to discover a possible use for 32 million ha of alfalfa. In 1998, this company started to produce 1,200 tons of alfalfa RuBisCO per year, at the same cost of producing soy protein. The purified RuBisCO, with a low degree of purification, is used in animal feed (rations) and in several areas of human nutrition - e.g., cookies. TNO - Innovation For Life is a Dutch company that also produces RuBisCO on a large scale, from sugar beet leaves. The process involves pressing, centrifuging and ultrafiltration, and has a production capacity of 10 kg protein / h, with a low degree of purification, around 35 to 40%. However, as mentioned earlier, these two companies do not produce RuBisCO in a purified state, as it is contaminated with other proteins and compounds from the plant leaves where it derives from.

Another Dutch company, NIZO, filled its RuBisCO purification process as a patent application in 2010. This process consists of protein extraction, with decantation and pressing, followed by purification by aggregation, precipitation and affinity separation, with final concentration by filtration and evaporation. The final product contains a minimum of 90% of the soluble RuBisCO and less than 0.1% (w/v) chlorophylls. However, the process is complex and has not yet been able to be scaled to an industrial level.

RuBisCO Foods is a Dutch company that produces protein gels and powders for the purpose of their application in food and feed. This company has developed and patented a unique technology that allows the extraction and purification of existing proteins in Lemna spp. (aquatic flowering plants). This plant has a significant growth rate, having the ability to double its biomass every 36 h, under optimum conditions, suitable for a daily harvest. Its rapid growth allows the production of 7 times more protein per ha of soil than soybeans. Therefore, this plant not only grows rapidly but also due to the fact that the plant is used entirely, i.e., not leaving any residues or waste. Unfortunately, the products manufactured by this company, gels and powders, have a green color, which indicates that the final product includes chlorophylls in its composition. In addition, RuBisCO in the final product is not pure at all. GreenProteins is a European project that presents as its main objective the production of high-quality proteins for later application in food. Proteins are extracted from sub-products produced by the food industry. This project, which began in 2016 and will end in 2021, was based on a new method for purifying RuBisCO from sugar beet leaves as described in US20150335043A1. Although the final product achieved is a white powder, which indicates the removal of chlorophylls, the described method uses organic solvents for the removal of chlorophylls, which is neither compatible with healthy food nor feasible for further application in the food industry. It is also important to note that, despite the fact that in the disclosure made to the public it is mentioned that RuBisCO is purified, the final product that is produced is the total extract of soluble proteins existing in the sugar beet leaves.

Summary of the invention

In a first broad aspect, the invention provides a process of obtaining a RuBisCO (ribulose- 1, 5-bisphosphate carboxyl ase/oxygenase) preparation from photosynthetic material, wherein the process comprises the steps of:

• lysing said photosynthetic material to extract RuBisCO accompanied by a fraction of chlorophylls of said photosynthetic material;

• performing precipitation with sodium phytate and/or calcium chloride;

• obtaining a phytate-calcium-RuBisCO insoluble ternary complex; and

• performing solubilization of said complex.

This process is particularly advantageous because, in preferred embodiments, it yields pure RuBisCO dissolved in water.

In preferred embodiments, the method is particularly advantageous in the obtention of pure RuBisCO which possess the following properties individually or in combination:

• Pure;

• Soluble (preferably readily soluble in water)

• Colourless

• Tasteless • Free from any toxic ingredients or their residues.

In a subsidiary aspect, said concentration of sodium phytate is of 5 to 20 mM; and optionally greater than 10 mM.

In a further subsidiary aspect, said concentration of calcium chloride is of 5 to 20 mM; and optionally greater than 10 mM.

In a further subsidiary aspect, said precipitation occurs at a low temperature; optionally said temperature being in a range of 2 to 8 degrees Celsius.

In a further subsidiary aspect, said solubilization step comprises the use of a glycine buffer.

In a further subsidiary aspect, said solubilization step comprises the use of a bicarbonate buffer or a carbonate buffer.

In a further subsidiary aspect, said solubilization step comprises the use of a buffer at a high pH; optionally said pH is greater than 10; optionally said pH is of 11.

In a further subsidiary aspect, said solubilization step comprises the use of one or any combination of any of the following: EDTA (ethylenediaminetetraacetic acid), calcium, calcium chloride, sodium chloride.

In a further subsidiary aspect, said process further comprises the step of removing phytate by filtration.

In a further subsidiary aspect, said process further comprises the step of removing phytate by one or a succession of microfiltration, ultrafiltration, or nanofiltration.

In a further subsidiary aspect, said process further comprises the step of removing water by reverse osmosis. In a further subsidiary aspect, the process further comprises the step of removing said buffer by ultrafiltration.

In a further subsidiary aspect, said step of ultrafiltration employs a molecular weight cut off membrane of 100 kDa or less.

In a further subsidiary aspect, said step of ultrafiltration employs a molecular weight cut off membrane of 300 kDa or less.

In a further broad aspect, the invention provides a process of obtaining a RuBisCO (ribulose-1, 5-bisphosphate carboxyl ase/oxygenase) preparation from photosynthetic material, wherein the process comprises the steps of:

• lysing said photosynthetic material to extract RuBisCO accompanied by a fraction of chlorophylls of said photosynthetic material; and

• performing precipitation by ethanol with concentrations of up to 15% (v/v).

In a subsidiary aspect, said ethanol concentrations are up to 5 % (v/v) or up to 10% (v/v).

In a further subsidiary aspect, the process comprises the steps of employing activated carbon to remove chlorophylls; whereby colourless, odourless and tasteless purified RuBisCO is obtained.

In a further subsidiary aspect, said activated carbon comprises bentonite.

In a preferred embodiment, said chlorophylls removal may be achieved by bentonite.

In a further subsidiary aspect, the process excludes any steps of addition of enzymatic inhibitors, reducing agents, denaturants, antioxidants, organic solvents (other than ethanol) or removers of phenolic compounds.

In a further broad aspect, the invention provides a RuBisCO preparation obtained by a process according to any one of the preceding aspects. In certain broad aspects, the process of purification involves one or more of the following steps: filtration (in a preferred embodiment ultrafiltration), differential centrifugation and selective precipitation. In preferred aspects, the process of purification comprises a step of precipitation with (i) 5% and (ii) 10% (v/v) ethanol, and with (iii) precipitation with sodium phytate and calcium chloride, with recoveries of 41%, 52% and

100% of RuBisCO, and 92%, 86% and 87% purity, respectively.

The results presented in the present application allowed the establishment of an efficient methodology to purify RuBisCO, which may easily be extended to an industrial scale suitable to be used for human consumption.

Aspects of embodiments of the invention provide an optimized method of purification for this protein which could overcome most, or all the disadvantages described above and simultaneously be regarded as GRAS (for ‘generally regarded as safe’). The final product was intended to be colourless, odourless, and tasteless, with the production methodology suitable to be extended to an industrial scale and used for animal and human consumption.

Exemplary methods are provided which take into consideration the biochemical characteristics of RuBisCO, the integrity of the protein and the need to expand to an industrial scale. Aspects of certain embodiments of the inventive method involve filtration (in a preferred embodiment ultrafiltration), differential centrifugation and selective precipitation.

In certain embodiments, the ultrafiltration method, using a molecular weight cut-off of 100 and 300 kDa were not very selective on their own, since it was possible to detect the presence of proteins with an apparent molecular mass higher than that of RuBisCO (ca. 550 kDa). The method involving ultracentrifugation in sucrose density gradients showed good purification, but low yields. The same was found for the selective precipitation at different pH values, mainly at pH 10, 11 and 12.

Preferred embodiments of the process of purification comprise a selective precipitation method step, with (i) 5% and/or (ii) 10% (v/v) ethanol, and/or with (iii) 15 mM sodium phytate and 15 mM calcium chloride, allowing a recovery of 41%, 52% and 100% total RuBisCO, and a purification of 92%, 86% and 87%, respectively. The purity of the isolated RuBisCO by these two methods was assessed by a very high sensitivity immunological procedure, immunoblotting, using polyclonal antibodies specific for the large subunit and for the small subunit of RuBisCO.

With the embodiment of a method using phytate and calcium, it is possible to obtain higher yields. However, it is a more complex methodology, comprising a greater number of steps in certain embodiments, including precipitation, filtration (in a preferred embodiment ultrafiltration) and solubilization. Removal of phytate from the final extract was preferred in certain embodiments, since phytate is considered by many authors as an anti-nutrient.

In a subsidiary aspect, the process of purification combines one or more of the method steps described herein. Advantageously, higher degrees of purification were achieved when precipitating first with 5% (v/v) ethanol and then with sodium phytate and calcium chloride, but with a lower yield. An alternative embodiment envisaged first precipitating with sodium phytate and calcium chloride and then precipitating with 5% (v/v) ethanol.

In a preferred embodiment, the process for purifying comprises a step of precipitation with phytate and calcium, followed by one or more steps of filtration (to remove phytate and calcium and/or remove calcium phytate and redissolution of the pure protein). In a preferred embodiment, the process employs ultrafiltration. In a further subsidiary aspect, the purified protein may be subjected to discoloration with activated charcoal, in order to remove pigments (e.g., chlorophylls and carotenoids) and therefore obtain a colorless extract.

In the end, a high-quality purification method for RuBisCO has been achieved, which is suitable for expansion to an industrial scale and used for human and animal consumption.

Brief description of the figures

Figure 1 shows quantification of RuBisCO and total soluble protein content in Lactuca sativa L. (lettuce), Eruca vesicaria L. (arugula), Brassica oleracea L. var. Acephala (Portuguese cabbage), Brassica oleracea L. var italica (broccoli), Spinacia oleracea L. (spinach), Nasturtium officinale L. (watercress), Spirulina sp., Coriandrum sativum L. (coriander) and Petroselinum crispum Mill (parsley) by the Bradford method (1976). For the quantification of RuBisCO, the samples were precipitated with 10% (w/v) PEG 6000. The values presented are the average of at least three biological replicates ± standard deviation. Figure 2 shows (A) Electrophoretic polypeptide profile of the total soluble protein from spinach leaves. The gel polypeptides were silver stained. (B) Representative image of the polypeptide profile of the protein fractions of spinach leaves obtained by SDS-PAGE, in acrylamide gel (17.5% w/v), supplemented with 10% (v/v) glycerol. Soluble spinach extract (T); RuBisCO precipitated with 10% (w/v) PEG 6000: (P) pellet; (S) supernatant. The gel polypeptides were stained with Coomassie Brilliant Blue G-250. In order to allow a qualitative analysis and comparison of the protein content, the T, P and S fractions were resuspended and loaded in the same volume, and the column of the T fraction was loaded with 15 pg of sample. LSU and SSU: Large and small subunits of RuBisCO, respectively. Molecular masses of standards are indicated in kDa.

Figure 3 shows example of an electrophoretic analysis by SDS-PAGE, of the different fractions, precipitated (P) and supernatant (S), of the total spinach leaf soluble protein extract precipitated with different ethanol concentrations. The gel contained 17.5% (w/v) acrylamide, was supplemented with 10% (v/v) glycerol, and polypeptides were stained with CBB-R-250. The soluble fractions were precipitated with 90% (v/v) ethanol before SDS-

PAGE analysis. All precipitated samples were resuspended at the same final volume and loaded at the same volume in the gel in order to obtain a comparative analysis. Thus, each column contains different amounts of protein: 2.5% (v/v) ethanol: S- 12 pg and P- 3 pg; 5%: S- 9 pg and P- 7 pg; 10%: S- 6 pg and P- 9 pg; 15%: S-8 pg and P- 7 pg; 20%: S-5 pg and P- 10 pg; 30%: S-4 pg and P- 11 pg; 40%: S-5 pg and P- 10 pg; 50%: S-4 pg and P- 11 pg; 60%: S- 1 pg and P- 15 pg; 70%: S- 0 pg and P- 15 pg; 80%: S-0 pg and P- 15 pg; 90%: S- 0 pg and P- 15 pg. LSU and SSU: large and small subunits of RuBisCO, respectively.

Figure 4 shows quantification of the proportion of RuBisCO and of the other proteins present in the precipitate obtained with different ethanol concentrations. The values are the average of at least three replicates ± standard deviation. Figure 5 shows an example of an SDS-PAGE electrophoretic analysis of the fractions obtained, pellet (P) and supernatant (S), after incubation of the total extract of spinach leaves with 10 mM sodium phytate and 10 mM calcium chloride, for 10 min at 42 ° C. The gel contained 17.5% (w/v) acrylamide, supplemented with 10% (v/v) glycerol. The soluble fraction was precipitated with 80% (v/v) acetone and resuspended in the same volume as the precipitate with phytate. Polypeptides in the gel were stained with CBB-R-250. Each column contains 10 pg protein. LSU and SSU: RuBisCO's large and small subunits, respectively. Figure 6 shows an example of an electrophoretic analysis by SDS-PAGE, of the fractions obtained, pellet (P) and supernatant (S), after incubation of the total extract of spinach leaves with different concentrations of sodium phytate and calcium chloride (5 mM, 15 mM and 20 mM), for 5 and 10 min, at 42 ° C. The soluble fraction was precipitated with 80% (v/v) acetone and dissolved in the same volume of sample buffer as that precipitated with phytate + calcium. The gel contained 17.5% (w/v) acrylamide, supplemented with 10% (v/v) glycerol. Polypeptides were stained with Coomassie G. Each fraction contains: 5 mM and 5 min: S- 10 pg and P- 10 pg; 5 mM and 10 min: S- 10 pg and P- 10 pg; 15 mM and 5 min: S- 5 pg and P- 10 pg; 15 mM and 10 min: S- 4 pg and P- 11 pg; 20 mM and 5 min: S- 2 pg and P- 13 pg; 20 mM and 10 min: S- 0 pg and P- 15 pg. LSU and SSU: RuBisCO's large and small subunits, respectively.

Figure 7 shows an example of an SDS-PAGE electrophoretic analysis of the fractions obtained, precipitate (P) and supernatant (S), after incubation of the total extract of spinach leaves with 15 mM sodium phytate and 15 mM calcium chloride, during 10 min at different temperatures, 4 °C, 25 °C and 42 °C. A 17.5% (w/v) acrylamide gel was used, supplemented with 10% (v/v) glycerol. The polypeptides were stained with Coomassie G. The soluble fraction was precipitated with 80% (v/v) acetone and diluted to the same volume as that precipitated with phytate and calcium, thus the gel columns were loaded with the same sample volume, to allow a comparative analysis, corresponding to: 4 °C: S- 4 pg and P- 15 pg; 25 °C: S- 2 pg and P- 12 pg; 42 °C: S- 4 pg and P- 11 pg. LSU and SSU: RuBisCO's large and small subunits, respectively. Figure 8 shows quantification of the proportion of RuBisCO and of the remaining proteins of the precipitate and respective supernatant obtained after incubation of the total extract of spinach leaves with 15 mM sodium phytate and 15 mM calcium chloride, for 10 min at 4 °C. Values are the average of at least three replicates ± standard deviation.

Figure 9 shows an example of an SDS-PAGE electrophoretic analysis of the fractions obtained, precipitate (P) and supernatant (S), after incubation of the total extract of spinach leaves with 15 mM sodium phytate and 15 mM calcium chloride, for 10 min at 4 °C and resuspension with different concentrations of EDTA. The gel contained 17.5% (w/v) acrylamide, supplemented with 10% (v/v) glycerol. The gel polypeptides were stained with CBB-R-250. The soluble fractions were precipitated with 80% (v/v) acetone. Each fraction contained 0 pg of protein for supernatants and 15 pg for precipitates. LSU and SSU: RuBisCO's large and small subunits, respectively. Figure 10 shows an example of an SDS-PAGE electrophoretic analysis of the fractions obtained, precipitated (P) and supernatant (S), after incubation of the total extract of spinach leaves with 15 mM sodium phytate and 15 mM calcium chloride, for 10 min at 4 °C and resuspension with different concentrations of CaC12. The gel contained 17.5% (w/v) acrylamide, supplemented with 10% (v/v) glycerol. The gel polypeptides were stained with CBB-R-250. The soluble fractions were precipitated with 80% (v/v) acetone. Fraction S was loaded with 0 pg and fraction P contained 15 pg protein. LSU and SSU: RuBisCO's large and small subunits, respectively.

Figure 11 shows an example of an electrophoretic analysis by SDS-PAGE of the fractions obtained, precipitated (P) and supernatant (S), after incubation of the total extract of spinach leaves with 15 mM sodium phytate and 15 mM calcium chloride, for 10 min at 4 °C, followed by resuspension with different concentrations of EDTA, NaCl and a change in the pH value of the solubilization solution. The gel contained 17.5% (w/v) acrylamide, supplemented with 10% (v/v) glycerol. The gel polypeptides were stained with Coomassie G. The soluble fraction was precipitated with 80% (v/v) acetone. All samples were resuspended in the same volume of sample buffer, the same volume being loaded on the gel to allow a comparative analysis, corresponding to: A: S- 0 pg and P- 15 pg; B: S- 0 pg and P- 15 pg; C: S- 0 pg and P- 15 pg; D: S- 8 pg and P- 8 pg; E: S- 6 pg and P- 6 pg. LSU and SSU: RuBisCO's large and small subunits, respectively.

Figure 12 shows an example of an electrophoretic analysis by SDS-PAGE of the fractions obtained, precipitate (P) and supernatant (S), after incubation of the total extract of spinach leaves with 15 mM sodium phytate and 15 mM calcium chloride during 10 min at 4 °C and resuspension at different pH values (in 100 mM glycine buffer). The soluble fraction was precipitated with 80% (v/v) acetone and resuspended in the same volume of sample buffer as those precipitated by the method. The gel contained 17.5% (w/v) acrylamide, supplemented with 10% (v/v) glycerol. The gel polypeptides were stained with CBB-R- 250. Each column was loaded with the same volume, in order to allow a comparative analysis, corresponding to: pH 7.5: S- 0 pg and P- 15 pg; pH 8: S- 0 pg and P- 15 pg; pH 9; S- 2 pg and P- 10 pg; pH 10: S- 4 pg and P- 11 pg; pH 11: S-10 pg and P- 5 pg; pH 12: S- 3 pg and P- 2 pg; pH 13: S- 2 pg and P- 0 pg. LSU and SSU: RuBisCO's large and small subunits, respectively.

Figure 13 shows an example of an SDS-PAGE electrophoretic analysis of the fractions obtained, precipitated (P) and supernatant (S), after incubation of the total extract of spinach leaves with 15 mM sodium phytate and 15 mM calcium chloride, for 10 min at 4 °C, followed by three consecutive resuspensions of the same sample of precipitated RuBisCO with 100 mM glycine buffer, at pH 11 (identical results were subsequently obtained with 50 mM bicarbonate/carbonate buffer at the same pH). The gel contained 17.5% (w/v) acrylamide, supplemented with 10% (v/v) glycerol. The gel polypeptides were stained with Coomassie G. The soluble fractions were precipitated with 80% (v/v) acetone, dissolved and loaded in the same volume, corresponding to: Wash 1 : S- 20 pg and P- 5 pg; Wash 2: S- 1 pg and P- 3 pg; Wash 3: S- 3 pg and P- 0 pg. LSU and SSU: RuBisCO's large and small subunits, respectively.

Figure 14 shows the quantification of the proportion of protein obtained after incubation of the total extract of spinach leaves with 15 mM sodium phytate and 15 mM calcium chloride for 10 min at 4 °C, followed by solubilization of the precipitate with 100 mM glycine buffer at pH 11 (identical results were subsequently obtained with 50 mM bicarbonate/carbonate buffer at the same pH), in three successive washes. Values are the average of at least three replicates ± standard deviation.

Figure 15 shows the quantification of the total phytic acid content in the different stages of the precipitation of RuBisCO with sodium phytate and calcium chloride, by the colorimetric method described by Xu & Chang (2009). Values are the average of at least three replicates ± standard deviation. PSP- soluble part of the precipitation of RuBisCO with sodium phytate and calcium chloride; PP- precipitate obtained after precipitation of RuBisCO with sodium phytate and calcium chloride; PSS- soluble part obtained after protein solubilisation of PP; PS- precipitate obtained after protein solubilisation of PP; RU- retained from ultrafiltration through membranes with MWCO of 100 kDa.

Figure 16 shows lanes 1 to 3- Example of an electrophoretic analysis by SDS-PAGE of the fractions obtained after precipitation of RuBisCO with 5% (lane 1), 10% (lane 2) (v/v) ethanol from the total extract of spinach leaves and cheese whey (control: lane 3). The gel contained 17.5% (w/v) acrylamide, supplemented with 10% (v/v) glycerol. The gel polypeptides were stained with Coomassie G and each column contains 10 pg of protein. Lanes 4 to 6- Immunodetection of LSU in the precipitate obtained with 5% (v/v) ethanol (lane 4), 10% (lane 5) (v/v) ethanol and fermented mixture whey (control: lane 6), using anti-LSU antibody diluted 1: 5,000; each column contains 2 pg of protein. Lanes 7 to 9- Immunodetection of SSU in the precipitate obtained with 5% (v/v) ethanol (lane 7), 10% (lane 8) (v/v) ethanol and fermented mixture whey (lane 9), using anti-SSU antibody diluted 1: 5,000; each column contains 2 pg of protein.

Figure 17 shows lanes 1 and 2- Example of an electrophoretic analysis by SDS-PAGE, of the fractions obtained after precipitation of RuBisCO with 15 mM sodium phytate and 15 mM calcium chloride from the total extract of spinach leaves for 10 min at 4 °C, followed by resuspension with buffer 100 mM glycine at pH 11 (lane 1; identical results were subsequently obtained with 50 mM bicarbonate/carbonate buffer at the same pH) and cheese whey (control: lane 2). The gel contained 17.5% (w/v) acrylamide, supplemented with 10% (v/v) glycerol. The gel polypeptides were stained with Coomassie G and each column contains 10 pg protein. Lanes 3 and 4- LSU immunodetection in the sample obtained by precipitation with 15 mM sodium phytate and 15 mM calcium chloride, for 10 min at 4 ° C, followed by resuspension with 100 mM glycine buffer at pH 11 and ultrafiltration through the MWCO 100 kDa membrane (lane 3; identical results were subsequently obtained with 50 mM bicarbonate/carbonate buffer at the same pH) and fermented whey (lane 4), with anti-LSU antibody diluted 1: 5,000; each column contains 2 pg protein. Lanes 5 and 6- SSU immunodetection in the sample obtained by precipitation with 15 mM sodium phytate and 15 mM calcium chloride, for 10 min at 4 °C, followed by resuspension with 100 mM glycine buffer at pH 11 and ultrafiltered through a 100 kDa MWCO membrane (lane 5; identical results were subsequently obtained with 50 mM bicarbonate/carbonate buffer at the same pH) and fermented mixture whey (lane 6) with anti-SSU antibody diluted 1: 5,000; each column contains 2 pg protein.

Figure 18 shows a proportion of RuBisCO and other proteins present in the precipitated RuBisCO with the selected methods utilized individually: 5% and 10% (v/v) ethanol and 15 mM sodium phytate + 15 mM calcium chloride. Values are the average of at least three replicates ± standard deviation.

Figure 19 shows (A) Example of an SDS-PAGE electrophoretic analysis of the precipitate (P), obtained after incubation of the total extract of spinach leaves with 15 mM sodium phytate and 15 mM calcium chloride for 10 min at 4 °C and subsequent solubilization in 100 mM glycine buffer at pH 11 (identical results were subsequently obtained with 50 mM bicarbonate/carbonate buffer at the same pH), followed by precipitation with 5% (v/v) ethanol and analysis by SDS-PAGE on acrylamide gel (17.5% w/v), supplemented with 10% (v/v) glycerol. (B) Example of an electrophoretic analysis by SDS-PAGE, performed under the same conditions, but started with the precipitation with 5% (v/v) ethanol, and followed by the precipitation with 15 mM sodium phytate and 15 mM calcium chloride for 10 min at 4 °C. The soluble fractions were precipitated with 80% (v/v) acetone, and the samples dissolved in the same volume of buffer. The same volume was loaded on the gel, corresponding to: A: S- 10 pg and P- 2 pg; B: S- 0 pg and P- 4 pg. LSU and SSU: RuBisCO's large and small subunits, respectively. Figure 20 shows a sample of purified RuBisCO obtained by the precipitation method with sodium phytate and calcium chloride, before removal of chlorophylls by active carbon treatment (left) and after removal of chlorophylls by active carbon treatment (right).

Figure 21 shows freeze-dried samples of RuBisCO purified by the precipitation method with sodium phytate and calcium chloride after removal of chlorophylls by activated carbon.

Figure 22 shows a RuBisCO purification scheme 1 by the precipitation method with sodium phytate and calcium chloride.

Figure 23 shows a RuBisCO purification scheme 2 by the precipitation method with sodium phytate and calcium chloride.

Figure 24 shows a scheme of a possible industrial method of the laboratory method with sodium phytate and calcium chloride.

Detailed description

A selection of potential objectives

At least one objective of certain embodiments of this invention is to develop a suitable purification process for RuBisCO, appropriate to undergo scaling-up to an industrial scale and in accordance with the requirements of the food industry. During the first phase, RuBisCO was extracted from photosynthetic plants leaves and a cyanobacteria, using a precipitation method with PEG of the kind outlined in the prior art section, with the aim of quantifying the RuBisCO present.

Subsequently, several purification methods were tested that took into account the physical and chemical properties of the protein. Protein separation was performed by (i) ultrafiltration with molecular mass cut-offs (MWCO) of 300 kDa and 100 kDa; (ii) ultracentrifugation on sucrose density gradients; (iii) extraction at pH 11; (iv) precipitation at different pH values; (v) precipitation with different concentrations of ethanol; and (vi) precipitation with sodium phytate and calcium chloride. In the analysis of each method, several parameters were evaluated - the degree of purity of the protein obtained, yield, ease of execution, final solubilization and scale-increasing capacity.

To measure the degree of RuBisCO purity, an immunodetection technique was performed using polyclonal antibodies specific for each one of the RuBisCO subunits, with the objective of detecting the potential presence of smaller polypeptides which are fragments of the subunits formed during the purification protocols, as well as of protein contaminants. In addition, immunoblotting based on the use of a chemiluminescent substrate is an extremely sensitive technique, which does not show some of the lack of specificity problems associated with the ELISA technique.

METHODS

Plant materials used

The biological materials used to prepare embodiments of the invention were the leaves from spinach ( Spinacia oleracea L.), lettuce ( Lactuca sativa L.), arugula ( Eruca vesicaria L.), broccoli ( Brassica oleracea L., Italian variety), Portuguese cabbage ( Brassica oleracea L., variety Acephala), watercress (. Nasturtium officinale L.), parsley (. Petroselinum crispum Mill.) and coriander ( Coriandrum sativum L.), acquired in a local market. A photosynthetic microalga of the genus Spirulina belonging to phylum cyanobacteria was also used. The samples were washed, separated from the main vein, weighed and immediately frozen in liquid nitrogen, and stored at -20 °C until used.

As previously indicated, the application envisages the use of any photosynthetic material and preferably plant material. The term “plant” as used herein refers to an organism belonging to the kingdom Plantae. Examples of plants are not limited to those listed herein. These may include by way of illustration in particular trees, herbs, bushes, grasses, vines, ferns, mosses, and green algae.

Extraction of total soluble proteins In preferred embodiments, protein extractions may be performed at 4 °C in 100 mM Tris- HC1 (Tris(hydroxymethyl)aminomethane-hydrogen chloride) buffer pH 7.5, without the addition of enzymatic inhibitors, reducing agents, denaturants, antioxidants or removers of phenolic compounds, because the objective is to obtain a product suitable for a healthy food purpose.

Frozen leaves from various plant species employed may be ground in a porcelain mortar in the presence of liquid nitrogen until a fine powder is obtained, and in preferred embodiment, the proteins may be extracted in 100 mM Tris-HCl buffer, pH 7.5, in a ratio of 1 : 10 (w/v), with agitation, for 1 h to 4 °C. The homogenate may be subsequently centrifuged at 12,000 g in a Beckman Optima XL-90 ultracentrifuge for 1 h at 4 °C. The supernatant may be stored in aliquots at -20 °C until use.

Whilst the preceding envisages a particular embodiment, other method steps or processes are also envisaged within the scope of the invention for the breaking of the cellular membrane and wall of plants cells. Plant material may be subjected to mechanical, chemical and/or enzymatic lysis or other form of treatment as appropriate.

Separation of the lysed plant material into a liquid juice and a high solids slurry or a high solids cake may be achieved by any solid-liquid separation techniques known in the art. Examples of such separation techniques include sieving, filtration, centrifugation and decanting.

Precipitation with ethanol

In preferred embodiments, cold ethanol, previously cooled at for example -20 °C, may be added to the total leaf soluble protein extract until different final ethanol concentrations (2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% and 90% v/v) may be obtained. After a 4-h period incubation in ice and under constant agitation, each mixture may be centrifuged in a Beckman J2-21M/E centrifuge at 7,500 g for 15 min. The supernatant may be discarded, and the precipitate resuspended in Milli-Q water for further analysis. Precipitation with sodium phytate and calcium chloride a) Selective precipitation tests

No prior art purification method for RuBisCO, envisaged a specific precipitation with sodium phytate and calcium chloride.

To specifically precipitate RuBisCO from the leaf protein extract by adding sodium phytate and calcium chloride, in certain embodiments, the sample may be extracted in 50 mM Tris-HCl buffer pH 5.6 or pH 6.8, or simply in plain water, at a ratio of 1:10 (w/v). After extraction, different concentrations of sodium phytate and calcium chloride may be added (5, 10, 15 and 20 mM). Subsequently, the solution may be centrifuged at 16, 100 grin a Beckman J2-21M/E centrifuge for 10 min, and the supernatant and precipitate may be stored at -20 °C. b) Protein solubilization tests

The pellet obtained after precipitating RuBisCO with sodium phytate and calcium chloride may be relatively insoluble. For this reason, several embodiments provide methods for solubilizing it, using for example ethylenediaminotetraacetic acid (EDTA), CaCh, NaCl and different pH values:

Solubilization with EDTA

In certain embodiments, the RuBisCO precipitate obtained with sodium phytate and calcium chloride was resuspended in a solution with EDTA, which acts as a chelating agent, in the present case for Ca²⁺. RuBisCO suspensions were prepared, for different embodiments, with different EDTA concentrations (5, 10, 15, 30 and 50 mM) dissolved in 50 mM Tris-HCl buffer, pH 6.8, and were kept on ice for 2 h, under constant agitation, before centrifugation at 7,500 g in a Beckman J2-21M/E centrifuge for 10 min at 4 °C. The resulting precipitate was resuspended in water for further analysis and the supernatant placed in a "Vivaspin" tube (Sartorius stedim) and subjected to centrifugation at 3,000 g through a membrane with a MWCO of 100 kDa, to ensure phytate removal from the membrane-retained RuBisCO. Solubilization with calcium chloride

In certain embodiments, the RuBisCO precipitate obtained with phytate was dissolved in 50 mM Tris-HCl buffer, pH 6.8, containing different concentrations of calcium chloride (10, 15 and 30 mM). After 2 h on ice and under constant agitation, the sample was centrifuged at 7,500 g in a Beckman J2-21M/E centrifuge for 10 min at 4 °C. The supernatant was placed in a "Vivaspin" tube (Sartorius stedim) and subjected to centrifugation at 3,000 g- through a membrane with a MWCO of 100 kDa, to remove CaCh and residual phytate present in the extract containing the membrane-retained RuBisCO. RuBisCO precipitation with phytate and CaCh is dependent on the formation of the ternary insoluble complex RuBisCO-Ca²⁺-phytate, which requires balanced proportions of the three ingredients, much in the same way of the precipitation reaction between antigen and antibody or the insolubilization of the legume seed storage proteins in the presence of Ca²⁺ and Mg²⁺.

Solubilization with sodium chloride

In certain embodiments, the addition of a salt, namely of sodium chloride, aided in protein solubilization. Thus, a 50 mM Tris-HCl buffer solution was added to the RuBisCO precipitate obtained with phytate at pH 6.8 and pH 9.0, in the presence of 30 mM EDTA, containing different concentrations of sodium chloride (0.5, 1 and 2 M). The solution/suspension was kept under constant agitation on ice for approximately 2 h and then centrifuged at 7,500 g in a Beckman J2-21M/E centrifuge for 10 min at 4 °C.

Solubilization by changing the pH value of the solution

In certain embodiments, to resuspend the RuBisCO precipitate obtained with phytate at different pH values, a glycine-NaOH 100 mM buffer solution was used at different pH values (7.5, 8, 9, 10, 11, 12 and 13). Alternatively, a 50 mM bicarbonate/carbonate buffer was used. The precipitate was resuspended in each solution and placed at 4 °C for 2 h under constant agitation, and the solution was obtained by centrifugation at 7,500 g in a Beckman J2-21M/E centrifuge for 10 min at 4 °C. The solubilized sample was placed in a MWCO "Vivaspin" tube (Sartorius stedim) of 100 kDa and centrifuged in a Beckman Coulter Allegra TM 25R centrifuge at 3,000 g and 4 °C. The different fractions, both filtered and retained, were collected, and stored at -20 °C until used.

Quantification of phytic acid in purified RuBisCO samples

In certain embodiments, phytic acid was quantified following known colorimetric methods, in which a phytate extraction was performed for 16 h with 240 mM HC1. After incubation, the liquid extract was centrifuged at 7,500 g during 5 min, and the precipitate was discarded.

For the quantification of phytate, 0.1 mL of the supernatant was diluted with 2.9 mL distilled water. One mL of Wade reagent (0.03% w/v FeCb 6H₂O and 0.3% w/v sulfosalicylic acid) was added, and the mixture was stirred in a vortex and centrifuged at 14,000 grin a VWR centrifuge, Himac CT15RE, for 10 min at 10 °C. A standard curve was prepared, based on different concentrations of phytic acid (0, 5, 10, 20, 30, and 40 mg/mL). The absorbance was recorded at 500 nm in a spectrophotometer (Synergy HT, Bio-Tek), and the phytate values subsequently determined by linear regression.

Removal of the green colour and freeze-drying of purified RuBisCO

In certain embodiments, the isolated sample of purified RuBisCO was filtered in vacuum, through a layer of moist activated carbon, under a Whatman filter no. 1. Subsequently, the chlorophyll-free sample was frozen in liquid nitrogen and freeze-dried in a Micro Modulyo lyophilyzer coupled to a vacuum pump, model E2M2, from "Edwards", to obtain the final purified RuBisCO as a lyophilized powder.

RESULTS

Protein extraction

The extraction of proteins from plant leaves causes specific problems inherent to cell content, namely the presence of reducing agents, organic acids, phenolic compounds and terpenes, among others, and relatively high amounts of proteolytic enzymes. Many of these compounds interfere negatively with proteins, making it difficult or even blocking their subsequent study. In addition, the rupture of vacuoles makes the extraction medium quite acidic, which may induce the denaturation of many proteins and the increased activity of vacuolar proteolytic enzymes, impairing the obtaining of proteins in their native form and, consequently, their purification. In order to reduce this problem, certain embodiments of the invention perform protein extractions at low temperatures and in the presence of a buffer sufficiently strong to compensate for the decrease in the pH value. Preferred embodiments employ neither reducing agents nor protease inhibitors such as EDTA, phenylmethylsulfonil fluoride (PMSF), dithiotreitol (DTT) or agents to eliminate phenolic compounds such as polyvinylpolypyrrolide (PVPP) despite these being commonly used. Since preferred embodiments seek to provide methods of protein purification for food purposes, it was decided not to use any reagent that may be harmful to health, such as antioxidant compounds and protease inhibitors, most often used in conventional protein extractions, and it was therefore advantageous to work and conserve the samples at low temperatures, to avoid as much as possible proteolysis and denaturation phenomena. However, in preferred embodiments, a Tris-HCl 100 mM buffer at pH 7.5 was used to maintain neutral pH and minimize protein denaturation in the leaf extracts. Nevertheless, extraction with buffer at the pH values of 5.6 and 6.8, as well as extraction in plain water worked as well, provided that temperatures were kept low and the whole extraction procedure did not last long.

Selection of the photosynthetic biological material

The parameters used took into account the ease of obtaining the plant material, its ease of use in feeding, as well as the percentage of RuBisCO in the total soluble protein (Figure 1 and Table 1). In order to maximize the extraction of RuBisCO, a prior art method by PEG precipitation was initially tested.

The species Lactuca sativa L. (lettuce), Eruca vesicaria L. (arugula), Brassica oleracea L. cv Acephala (Portuguese cabbage), Brassica oleracea L. cv italica (broccoli), Spinacia oleracea L. (spinach), Nasturtium officinale L. (watercress), Spirulina sp., Coriandrum sativum L. (coriander) and Petroselinum crispum Mill (parsley) were selected, and healthy, ripe, green and healthy-looking leaves are used.

Figure 1 and Table 1 show the quantification of the total soluble protein of the leaves of each species and the relationship between the amount of RuBisCO and the other proteins. Table 1 shows RuBisCO content in relation to the total soluble protein in the different species, quantified by the Bradford method (1976), after precipitation with 10% (w/v) PEG 6000. The values presented are the average of at least three biological replicates ± standard deviation.

The results obtained in Figure 1 show that the species with the highest protein concentration was Spirulina sp., followed by Coriandrum sativum and Spinacia oleracea. However, with regard to the percentage amount of RuBisCO in relation to the total protein (Table 1), spinach was the species with the highest potential, having a lower percentage of other proteins, when compared to the other species. Thus, preferred embodiments of the invention have selected spinach for their methods.

Polypeptide profile of the selected photosynthetic biological material Spinacia oleracea was the chosen species, because it presented better purification potential, since approximately 70% of the total soluble protein of its healthy leaves is composed of RuBisCO, being necessary the removal of only ca. 30% of the remaining constituent proteins. First the characterization of the polypeptide profile of the total protein extract from spinach leaves was performed. The polypeptide profile is complex and corresponds to a wide range of molecular masses (15 to 150 kDa). The two most prominent polypeptide bands that are more representative (ca. 50 kDa and ca. 15 kDa) correspond to the subunits of RuBisCO (see Figure 2).

As can be seen in Figure 2B, lane P, RuBisCO's protein profile has two bands corresponding to LSU (~55 kDa) and SSU (~15 kDa) (Jensen, 1977). However, it is possible to observe polypeptides smaller than 55 kDa, possibly resulting from enzymatic hydrolysis of LSU. These bands, with a mass of 28, 37, 42 and 44 kDa, may come from oxidative stress or degradation from the N-terminal end to the residue of Lys 18, represented by a band with 53 kDa. Another band with 65 kDa can be equally formed by the covalent bond between an LSU and an SSU. Other bands will certainly correspond to polypeptides that have been co-purified with RuBisCO, thus representing contaminants. Due to its feasibility and efficiency, the PEG precipitation method was used as the standard method.

Tested methodologies for the purification and isolation of RuBisCO

Several RuBisCO purification tests were carried out, taking into account the physical and chemical properties of the protein, namely the isoelectric point, hydrophobicity, molecular mass, structure and resistance to denaturation. Taking into account the objective of improving a RuBisCO purification method for possible expansion at the industrial scale, for each method several parameters were evaluated, such as purity, yield, ease of execution, final solubilization of protein and scale-up capacity.

Two primary embodiments were identified, firstly involving RuBisCO precipitation with sodium phytate and calcium chloride; and secondly with precipitation of RuBisCO with 5% and 10% (v/v) ethanol.

Preferred embodiments were developed which involved, in addition to precipitation with sodium phytate and calcium chloride, further advantageous steps selected from: filtration (in certain embodiments microfiltration, ultrafiltration, nanofiltration or reverse osmosis) solubilisation at high pH values, and precipitation with ethanol.

Selective precipitation by ethanol

Figure 3 shows the results obtained with precipitation of RuBisCO with ethanol. At high concentrations of ethanol, RuBisCO precipitation was not selective, since almost all proteins precipitated. However, at low ethanol concentrations, namely 2.5%, 5%, 10% and 15% (v/v), it was possible to observe precipitation selectivity of RuBisCO.

In a preferred embodiment, the RuBisCO purification was obtained with the ethanol precipitation procedure with 2.5% (v/v) ethanol. However, the yield was relatively low (Figure 4 and Table 2), approximately 20% of the total soluble protein and 31% of the total RuBisCO. Also, with 5 and 10% (v/v) ethanol, a good purification was obtained, with higher yields, about 45 and 60% of the total protein and 53 and 67% of the total RuBisCO, respectively.

Table 2 shows a RuBisCO extraction yield in precipitate obtained with different ethanol concentrations. RuBisCO recovery yield values are expressed as a proportion of RuBisCO isolated in relation to the amount of RuBisCO in the original sample, with an average of at least three replicates ± standard deviation.

Ethanol precipitation of RuBisCO showed promising results with regard to precipitation with 5% and 10% (v/v) ethanol, reconciling a good purification in what selectivity is concerned with a good yield. However, the precipitate obtained showed a relatively low water solubility, being a negative aspect for its implementation in the food industry.

Precipitation with sodium phytate and calcium chloride

In certain embodiments, a method employing sodium phytate and calcium chloride was devised to isolate RuBisCO from a spinach leaf aqueous extract. The results obtained are shown in Figure 5.

By analyzing Figure 5, it is possible to conclude that the extraction yield of RuBisCO from the spinach leaf aqueous extract was less than 50%.

Preferred embodiments, seek to optimize the process, in order to obtain a higher yield and degree of precipitated RuBisCO. One approach was to determine the optimum concentrations of sodium phytate and calcium chloride required for the maximum precipitation of RuBisCO and also the time required to obtain a higher yield. To this end, different amounts of sodium phytate and calcium chloride were added to the total extract of spinach leaves, using different times, 5 and 10 min. Throughout the test, the temperature variable was maintained at 42 ° C (Figure 6).

The analysis of Figure 6 reveals that the higher the concentration of sodium phytate and calcium chloride, the greater RuBisCO precipitation and the lower the precipitation selectivity. Thus, the optimum concentration for both reagents was determined to be 15 mM. Regarding the incubation time, it was found that after 5 min no total precipitation from RuBisCO was achieved, with 10 min being chosen as the reaction time.

Once the incubation time and concentrations of sodium phytate and calcium chloride were selected, optimized to obtain the best RuBisCO purification, different reaction temperatures were tested, since no enzymatic proteolytic inhibitors were used in the protein extraction. Under these conditions, because the reaction at 42 °C could imply an intense proteolysis of RuBisCO, two temperatures, 4 °C and 25 °C, were tested, maintaining the temperature of 42 °C as a control (Figure 7).

Figure 7 shows that RuBisCO precipitation was much more efficient at 4 °C, obtaining a more selective precipitate than at other temperatures. Taking into account the results obtained, i.e. ca. 100% of the removal of RuBisCO (Figure 8 and Table 3), it was concluded that the most appropriate methodology for the specific precipitation of RuBisCO from the protein extract of spinach leaves was the addition of 15 mM sodium phytate and 15 mM calcium chloride, for 10 min with stirring, at 4 °C.

Table 3 shows the yield of the extraction of RuBisCO in the different fractions, precipitated and corresponding supernatant, obtained after incubation of the total extract of spinach leaves with 15 mM sodium phytate and 15 mM calcium chloride, for 10 min at 4 °C. RuBisCO's recovery yield values are expressed as a percentage of RuBisCO precipitated in relation to total RuBisCO in the original sample, with an average of at least three replicates ± standard deviation.

With this methodology, a precipitate composed of protein was obtained, essentially composed of RuBisCO, but strongly linked to phytate and calcium. The next step was preferably solubilizing the precipitated RuBisCO.

Solubilisation of RuBisCO precipitated with sodium phytate and calcium chloride

Precipitation of RuBisCO with sodium phytate and calcium chloride resulted in a precipitate insoluble in water, once the phytate-calcium-RuBisCO ternary complex was formed. It was therefore necessary, in a subsequent phase, to solubilize the protein and remove the phytate and calcium present. In this sense, during this part of the experimental work, several attempts of solubilization were made, with the evaluation of the most effective method based on its performance. It was considered important to remove phytate because it is considered an anti-nutrient since, when ingested in excess, it decreases the bioavailability of several essential minerals. Embodiments of the inventive method include solubilization of the protein with one or more of the following: EDTA, calcium, EDTA + sodium chloride and alteration of the pH of the solubilization solution. Since EDTA has a chelating capacity, it was expected that it would bind to calcium, thus breaking the existing complex phytate-calcium-RuBisCO and releasing RuBisCO (Figure 9). This depends, of course, on the relative affinities of EDTA and phytate for calcium. Regarding calcium (Figure 10), an excess of this element could compete with the protein for binding to phytate. With this assay it was possible to understand that the protein binds to phytate through calcium and that phytate exhibits a greater affinity for calcium than EDTA (Figures 9 and 10).

The results obtained indicated that treatment of the phytate-calcium-RuBisCO precipitated complex with EDTA or calcium did not allow the solubilization of the precipitated RuBisCO. In this sense, small amounts of sodium chloride (NaCl) were added, which can facilitate protein solubilization. These results, shown in Figure 11, also did not produce RuBisCO solubilization, when 0.5 M, 1 M or 2 M sodium chloride was added, together with 30 mM EDTA. However, when these treatments were performed after changing the pH of the solution, some degree of solubilization was achieved.

In a preferred embodiment, solubilization of the protein was observed after changing the pH (Figure 11 D, E) and since the initial binding of phytate, calcium and protein is dependent on a pH between 4.8 and 10.4, higher pH values of the solubilizing solution were tested in an attempt to break the bonds between RuBisCO and phytate, via calcium. Thus, protein solubilization was tested only by changing the pH value of the solubilizing solution (i.e. in the absence of EDTA, extra calcium and sodium chloride), in which a 0.1 M glycine buffer solution was used, which has a buffering power at alkaline pH values and can be used in the food industry. The results are shown in Figure 12. Alternatively, identical results were obtained with 50 mM bicarbonate/carbonate buffer, pH 11.

The electrophoretic profile shows that the best solubilization was achieved at pH 11. However, total solubilization was not achieved. At pH 12 and pH 13 there was a loss of protein, with studies that mention the existence of proteases capable of acting at alkaline pH values. As the process was carried out at 4 °C, this should not have happened. Another explanation may be the very high pH value, which may have induced the alkaline hydrolysis of some proteins, breaking them down into their constituent amino acids, or simply protein denaturation.

In order to achieve a greater solubilization of the precipitated RuBisCO, several successive washes were carried out with buffer at pH 11. Through the analysis of Figure 13 it is possible to verify that total solubilization of precipitated RuBisCO is achieved after three washes, ceasing to exist protein in the precipitate.

Solubilization with successive washes at pH 11 revealed a total solubilization of the precipitated isolated RuBisCO, with a yield of approximately 100% solubilisation (Figure 14).

The extraction yield of RuBisCO in relation to the total soluble protein of spinach leaves was approximately 100% (Table 3), being higher than that achieved with the ethanol precipitation method. The precipitation method with sodium phytate and calcium chloride proved, therefore, to be a good method of purification of RuBisCO, having achieved a good degree of purification, excellent yield and a final soluble sample.

Quantification of phytic acid in the purified RuBisCO fraction

After precipitation of RuBisCO with sodium phytate and calcium chloride and subsequent solubilization of the precipitated protein with 100 mM glycine buffer or with 50 mM bicarbonate/carbonate buffer, both at pH 11, a green-colored solution with protein and free phytate and a white precipitate containing phytate complexed with calcium was obtained. Thus, it was necessary to remove the soluble phytate present in the sample since, as previously mentioned, it is related to several anti -nutritional characteristics when ingested in excess. It was decided to carry out an ultrafiltration through 100 kDa MWCO membranes (or, alternatively, microfiltration, nanofiltration or reverse osmosis), so that the phytate present could be removed and allowing, simultaneously, desalting and protein concentration steps, since RuBisCO is retained by a membrane with MWCO of 100 kDa.

During ultrafiltration, several washes were performed with water, in an attempt to promote the complete removal of phytate and also of the buffer solution, as well as of any other small molecules that may be present, while obtaining RuBisCO solubilized in water. Determination of phytic acid content, both in the filtrate and in the RuBisCO fraction, was carried out according to prior art colorimetric methods, with the result being 0 g/mL (±0.006 g/mL) in the RuBisCO fraction alone (Figure 15). Ultrafiltration had the advantage of, in addition to removing phytic acid, also removing some proteins that may be present in the purified RuBisCO sample, especially those with molecular masses of less than 50 kDa, as shown by tests performed with membranes with a MWCO of 100 kDa.

In short, through the use of 100 kDa MWCO membranes, complete removal of phytic acid from the purified RuBisCO sample was achieved. In this way, it was possible to obtain a product that is safe for human consumption and suitable for use in the food industry.

Analysis of the degree of purity of RuBisCO

The methods identified for optimisation for the industrial purification of RuBisCO were precipitation with 5% and 10% (v/v) ethanol and precipitation with 15 mM sodium phytate and 15 mM calcium chloride for 10 min at 4 °C.

In order to confirm the contamination level of RuBisCO in the different methods, a highly sensitive immunodetection using a chemiluminescent substrate was performed with specific antibodies against the large subunit (anti-LSU) or against the small subunit (anti- SSU) of RuBisCO (Figures 16 and 17). The final result of immunodetecting a protein antigen by an antibody depends, among other factors, on the amount of antigen present and on the dilution of the antibody used. The immunoblotting conditions have been previously optimized for the same antibodies and antigens, to achieve great accuracy and sensitivity (Esquivel, 1995).

Regarding the precipitation with 10% (v/v) ethanol, there was a partial degradation of the LSU, which resulted in a polypeptide with a molecular mass slightly below 55 kDa, which may correspond to the 53 kDa polypeptide (Figure 16).

Likewise, in the immunodetection performed for the sodium phytate and calcium chloride method, it was possible to find a degradation of the large subunit, resulting in a polypeptide of approximately 53 kDa (Figure 17). However, what stood out the most was the polypeptide obtained by the covalent bond between an SSU and an LSU, which gave rise to a band with 65 kDa, called P65.

Comparing the gels stained with Coomassie with the immunoblots, it is possible to conclude that the precipitation with 5% (v/v) ethanol was the one that presented greater selectivity for RuBisCO, originating a smaller number of contaminating polypeptide bands, which were not originated by degradation of the enzyme subunits (i.e., were not degradation products of LSU and SSU).

Calculation of the degree of purification of RuBisCO obtained by the different methods utilized individually was carried out by densitometry and showed that 92% of the RuBisCO was found in the precipitate obtained with 5% (v/v) ethanol, 86% in the precipitate obtained with 10% (v/v) ethanol, and in 87% in the precipitate obtained with 15 mM sodium phytate and 15 mM calcium chloride. With this analysis it was possible to verify that a good purification of RuBisCO was achieved with both methods (Figure 18 and Table 4).

Table 4 shows the degree of purification of RuBisCO obtained with the selected methods utilized individually: 5% and 10% (v/v) ethanol and 15 mM sodium phytate + 15 mM calcium chloride. The values of the degree of purification of RuBisCO are expressed as a percentage of RuBisCO alone in relation to the amount of RuBisCO in the purified sample and are the average of at least three replicates ± standard deviation.

Based on the results obtained, it was possible to conclude that the ethanol precipitation method showed the potential to purify RuBisCO, since it obtained a good selectivity in precipitation but an average yield, achieving only 41% of the total RuBisCO present in the spinach leaves (4.41 mg / g fresh weight) with 5% (v/v) ethanol and 52% (5.83 mg / g fresh weight) with 10% (v/v) ethanol (Figure 4). This method can also be easily expanded to an industrial scale, due to the reduced number of steps and simplicity of the process. However, it presents the aforementioned disadvantage of the difficulty in solubilizing ethanol-precipitated RuBisCO.

Regarding the precipitation method with sodium phytate + calcium chloride, it proved to be a good method of purification, since a 100% yield was achieved, that is, there was a total removal of RuBisCO from the total extract of spinach leaves (7.88 mg / g fresh weight). The final sample was shown to be totally soluble and 87% pure. Chlorophyll removal from the RuBisCO purified sample

Activated carbon is a porous material formed by carbon atoms, which has the ability to adsorb molecules, through the pores and cavities that have the capacity to establish intense Van der Waals interactions. This is used, for example, for medicinal purposes, in people who suffer from intestinal problems, as well as in the oil industry as a method of dechlorination.

This activated carbon processing was performed only with RuBisCO purified with the precipitation method with sodium phytate and sodium chloride (Figures 20 and 21). The subsequent freeze-drying allowed a white and soluble powder to be obtained.

Several other substances were tested with various degrees of success in the removal of chlorophylls from purified RuBisCO samples. One of the best ones involved the use of bentonite.

Embodiments of industrial isolation methods for RuBisCO

Two purification schemes are presented, in which precipitation, solubilization and ultrafiltration were performed in figures 22 and 23.

In the embodiment of figure 22, the first step is the selection of an appropriate photosynthetic material. The invention is not limited to any particular species. Nevertheless, spinach leaves have provided a particularly efficient process as compared to other species.

In preferred embodiments, the process involves the step of selecting aquatic higher plants (e.g., duckweed / Lemna minor) or algae (e.g., macroalgae) which:

- can grow in tanks and may have mass doubling times as little as 36 h under optimum conditions;

- can grow all year round (with much longer mass doubling times during winter) and produce as much as seven times (in the case of duckweed; or even more in the case of certain micro or macroalgae) the amount of protein a year per unit area when compared to a field of soybeans;

- occupy a minimum area of land when compared to traditional farming;

- do not pollute the environment (freshwater plants);

- contribute to carbon dioxide retention; - are used entirely, i.e. do not leave any residues or waste;

- allow for the selection of the highest RuBisCO content-species;

- require nothing else than sunlight and a mineral solution.

Another embodiment envisages improving the RuBisCO yield by hydroponic cultivation.

A process step of lysing said photosynthetic material is followed to extract RuBisCO accompanied by chlorophylls. A preferred embodiment employs a 50 mM Tris- HC1 (Tris(hydroxymethyl)aminomethane-hydrogen chloride) buffer, pH 5.6 or 6.8. Performing the extraction at pH 5.6 was initially selected with the aim of subsequently extracting RuBisCO and chlorophylls from the photosynthetic material in plain water.

The following alternative to Tris-HCl are envisaged water, as well as phosphate, citrate, succinate, and acetate buffers. The pH of said buffer is preferably with the following range of 5 to 9. The concentration of said buffer is preferably within the range of 0 to 100 mM.

The pH level of said extraction step is preferably within the range of pH 5 and pH 9. Most preferably, the pH is of 5.5 to 7.5. Advantageously, the pH is of 5.6 or 6.8.

The duration of the extraction process of the soluble protein is within the range of several minutes to 2 hours, preferably taking place at low temperatures (between 0 degrees Celsius and 4 degrees Celsius).

Upon completion of the extraction step, the liquid is conveyed into a further station in which it is mixed with sodium phytate and calcium chloride in order to lead to the precipitation of RuBisCO.

Bentonite may be used as an alternative to phytate. Other divalent cations (eg. magnesium) may also be used as an alternative to calcium.

In a preferred embodiment, the selective precipitation of RuBisCO from a total soluble protein extract obtained from a photosynthetic cell, tissue, organ or organism relies on a specific proportion of three ingredients: • Sodium phytate

• RuBisCO

• Calcium chloride to allow formation of the insoluble ternary complex phytate-calcium-RuBisCO, which removes RuBisCO from solution, therefore separating it from the other soluble proteins.

To achieve total precipitation of RuBisCO and recovery of all RuBisCO in the insoluble ternary complex, the following proportions were used under the optimized conditions described in the present application: sodium phytate: 15 mM, RuBisCO: 15 mL of the extract, and calcium chloride: 15 mM.

In certain embodiments, times and temperatures may vary from minutes to several hours and from 0 to 40 degrees Celsius. In a preferred embodiment, the duration was of 2 hours and the temperature range was of 0 to 4 degrees Celsius.

In a preferred embodiment, the concentration of sodium phytate is of 15 mM and the concentration of calcium chloride is of 15 mM. The duration of this step is typically of no more than 120 minutes, no more than 90 minutes, no more than 60 minutes, no more than 30 minutes, no more than 15 minutes, no more than 10 minutes.

The temperature for the precipitation is preferably maintained at no more than 10 degrees, no more than 7 degrees, no more than 5 degrees Celsius. Preferably, the mixture is maintained at approximately 4 degrees Celsius.

Once the precipitation step is complete, the contents are submitted to centrifugation or other filtration means. For best results (including speeding up the whole process), a typical centrifugation will be carried out at 10,000 to 50,000 g and at 0 to 4 degrees Celsius during 10 minutes.

One of the products of the centrifugation is an insoluble ternary complex of phytate- calcium-RuBisCO in the form of pellet. The RuBisCO present in the resulting pellet is then subjected to solubilization.

In a preferred embodiment, the solubilization process employs glycine buffer. In an alternative embodiment, a bicarbonate-carbonate buffer system may be used with similar efficiency.

In a preferred embodiment, the buffer has a concentration of 50 to 100 mM and a pH of 11. In preferred embodiment, concentrations of 10 to 100 mM may be used.

The duration of the RuBisCO solubilization step is typically of no more than 120 minutes, no more than 90 minutes, no more than 60 minutes, no more than 30 minutes, no more than 15 minutes, no more than 10 minutes.

The temperature for the RuBisCO solubilization is preferably maintained at no more than 10 degrees, no more than 7 degrees, no more than 5 degrees Celsius. Preferably, the mixture is maintained at approximately 4 degrees Celsius.

The resulting product is submitted to a further centrifugation stage or filtration stage. For best results (including speeding up the whole process), a typical centrifugation will be carried out at 5,000 to 20,000 g and at 0 to 4 °C during 10 min.

The centrifugation results in the calcium-phytate being removed as a pellet. The pellet may be subjected to one or more further cycles of solubilization in glycine buffer or in a bicarbonate-carbonate buffer system.

A further centrifugation stage may then be pursued with the resulting RuBisCO- containing supernatant liquid, whereby further calcium-phytate is removed as a pellet. This pellet may be subjected once more to further cycles of solubilization in glycine buffer or in a bicarbonate-carbonate buffer system.

The resulting supernatant is then subjected to filtration and preferably to ultrafiltration through a 100 kDa MWCO membrane. Whilst a molecular weight cut-off membrane of 100 kDa or less is preferred, the following other membranes are envisaged membranes with MWCO between 100 and 300 kDa.

In further embodiments, other small-pore ultrafiltration membranes, as well as nanofiltration membranes may be used.

The retained larger proteins will now almost solely be RuBisCO and may be subjected to an activated charcoal or activated carbon stage for the removal of chlorophylls.

To avoid loss of RuBisCO, a suitable amount of activated charcoal must be used. Higher amounts of activated charcoal will lead to loss of RuBisCO. In a preferred embodiment, 19 g of activated charcoal may be used per litre of water-dissolved RuBisCO. In a preferred embodiment, less than 50 g of activated charcoal may be used per litre of water-dissolved RuBisCO.

Bentonite may be used as an alternative to activated charcoal.

The resulting product is then colourless, odourless and tasteless purified RuBisCO.

Figure 23 shows a further embodiment where a photosynthetic material is selected.

It may for example be frozen photosynthetic material. The frozen photosynthetic material may be ground to a fine powder. In one embodiment, the photosynthetic material may be or include spinach leaves.

The first step of lysing may be performed in a 50 mM Tris-HCl buffer at pH 5.6 or in plain water.

It may be accompanied or followed by slight agitation. Thus allowing potentially the combination of chemical and mechanical lysis. The slight agitation may be applied for a defined period of no more than 3 hours, no more than 2 hours, no more than 1.5 hours. Preferably, the agitation may be applied for 1 hour. The temperature of the extraction process may be maintained at around 8 degrees Celsius. Optionally, the temperature may be less than 10 degrees Celsius but no less than 2 degrees Celsius. A preferred range may be of 4 to 8 degrees Celsius. Following the extraction step, the liquid may be subject to a centrifugation or other filtration stage. The centrifuge may be performed at 12,100 g. For best results, the first extract obtained from the photosynthetic material must be centrifuged at 5,000 to 50,000 g.

The duration of centrifugation may be of no more than 2 hours but preferably around

1 hour.

The resulting supernatant (15 mL) which will contain the total soluble proteins may then be incubated in the presence of suitable amounts of sodium phytate (15 mM) and calcium chloride (15 mM) and stirred. The stirring step may be preferably of 2 hours at a temperature of 4 degrees. Unlike the relative proportions of sodium phytate, RuBisCO and calcium chloride, which must be precise to maximise RuBisCO precipitation, those values may vary widely. Therefore, the duration of the stirring step may span from several tens of minutes to several hours, at a temperature of 2 to 8 degrees Celsius. A further step of centrifugation then follows which may be at 16,100 g for 10 minutes. In preferred embodiments, the centrifugation may be carried out at 10,000 to 50,000 g and at 0 to 4 degrees Celsius during 10 minutes. The resulting supernatant contains the remaining soluble proteins whilst the pellet contains the ternary insoluble complex of phytate-calcium RuBisCO.

The pellet or pellets may then be subjected to a solubilization process which may be of the kind described with reference to figure 22.

This may involve the provision of a buffer of glycine. The concentration of glycine may be of 100 mM. The pH of the solution may be of 11. Alternatively, 50 or 100 mM bicarbonate-carbonate buffer at a pH 11 may be used.

After a first stage of solubilization, the resultant pellet and supernatant may be subjected to one or more centrifugations. The preferred rating for the centrifuge may be of 7,500 g for 10 minutes.

The products of the centrifugation stage are respectively a supernatant of RuBisCO and a pellet of calcium phytate.

The supernatant of RuBisCO may then be subjected to filtration, ultrafiltration or nanofiltration. In a preferred embodiment, the ultrafiltration employs a molecular weight cut-off membrane of 100 kDa or less. Furthermore, the resultant passes through an activated carbon which may include activated charcoal in order to yield once more the colorless, odorless and tasteless purified RuBisCO.

Figure 24 shows a further embodiment which may be at a very large industrial scale. Whilst this description illustrates a number of preferred steps, further steps may be required as appropriate. These are provided for illustration purposes.

The extraction process may involve large scale containers or cylinders in which chemical and/or mechanical lysing may take place for the photosynthetic material which may be selected as appropriate. An appropriate solvent may be provided such as Tris-HCl buffer or simply plain water. The pH may be selected to be 5.6 to 6.8 pH units. The process may be assisted by agitation and other mechanical means as appropriate.

Once the resultant from the extraction process is obtained, the resultant may flow through conventional means to a precipitation stage which may involve known cylinders in which sodium phytate and calcium chloride may be provided.

In a preferred embodiment to achieve the total precipitation of RuBisCO and recovery of all RuBisCO in the insoluble ternary complex, the following proportions are used under the optimized conditions described herein:

• Sodium phytate: 15 mM;

• RuBisCO: 15 mL of the extract;

• Calcium chloride: 15 mM.

A number of filtration stages may be provided. At this stage filtration may be obtained through filter membranes of a large pore size, using particle filtration or microfiltration.

The resultant of the filtration steps may be either discarded for further processing or retained for solubilization by adding appropriate quantities of buffer such as glycine buffer at 100 mM, 50 mM bicarbonate/carbonate buffer or any buffer at a sufficient buffering capacity around the pH value required, all at a pH close to 11.

A number of filtration stages may be provided which may include one or more centrifugations. In certain embodiments, for best results (including speeding up the whole process), a typical centrifugation will be carried out at 5,000 to 20,000 g and at 0 to 4 degrees Celsius during 10 minutes.

The main resultant from the filtration/centrifugation process is a supernatant which primarily contains RuBisCO and chlorophylls whilst pellets of calcium phytate are produced.

The supernatant may then be subjected to ultrafiltration.

This last filtration stage may be performed by several means and with different objectives due to very large size of RuBisCO (ca. 550 kDa). The main objectives may be, depending on the type and pore of the selected membrane: (i) desalting RuBisCO and obtain the enzyme dissolved in water, (ii) concentrating RuBisCO by the removal of water, and (iii) removing ions, metabolites, peptides and even small proteins or protein fragments that may be present in residual amounts. In this respect, microfiltration, ultrafiltration, nanofiltration or reverse osmosis may be used.

The retained resultant is then subjected to activated carbon which may take the form of activated charcoal. At appropriate concentrations, the activated charcoal may capture all the chlorophylls, but none of the RuBisCO.

The resultant liquid is then pure from chlorophylls and 100% RuBisCO. Steps of either spray-drying or freeze-drying would yield a powder of colourless, odourless, and tasteless of pure RuBisCO.

Embodiments of the process at an industrial scale may involve some additional steps, namely the recovery of precipitates that can be resolved, for example, by a filter system.

The approximate amount of plant material and reagents required for the purification of 1 kg of RuBisCO alone, are shown in Table 5. The applicants have already obtained more favourable results during an optimization process.

Table 5 shows the estimation of the amount of plant material and reagents required for the purification of 1 kg of colourless, odourless and tasteless pure powdery RuBisCO.

The cost of sodium phytate is high, contributing to an equally high production cost. Added to these costs are the costs of the two buffers used, Tris-HCl and glycine, the precipitate recovery filters, the ultrafiltration membranes, the activated carbon and the maintenance of all the equipment. To avoid high reagent costs, however, one can find alternatives processes, namely the recycling / reuse of sodium phytate and calcium chloride, sell calcium phytate (a by-product of this RuBisCO purification procedure, after solubilization of the precipitated RuBisCO) which has a high commercial value, or the acquisition of phytate without a very high degree of purity, which would considerably lower the price of the final product. In addition, at an industrial scale, the total soluble protein from spinach (or from any other photosynthetic tissues or cells) may well be extracted in water. In preferred embodiments, Tris-HCl and glycine may be substituted by plain water and bicarbonate/carbonate buffer, respectively.

The approach/protocol presented in the present invention to purify RuBisCO as a premium protein for human and animal consumption is different from the procedures described in the prior art for at least one of the following reasons:

- It comprises a short protocol containing few steps, suitable to be scaled-up to an industrial level and producing the pure enzyme at a reasonable price.

- It does not use organic solvents (with the possible exception of ethanol) or any other reagents or solvents which may be harmful to human health.

- Chlorophylls are removed, which means that the final product is a white powder, not a green one. - As RuBisCO is often found already at a ‘purification degree’ of 50% or more in the leaves of C3 plants, most other approaches are only based on a simple total soluble protein extraction (thus including many thousands of other different proteins, which may include allergenic as well as other toxic proteins) but otherwise claim that their RuBisCO is pure. - The method described in the present invention allows the purification of RuBisCO with a high degree of purity and yield.

- The final product obtained is white powder, without odour or taste.

- Lastly and maybe most importantly, many prior art procedures described for the large scale purification of RuBisCO yield an insoluble protein; by contrast, embodiments of the invention provide a soluble pure form of RuBisCO.

The final product obtained after application of the present invention to RuBisCO purification is a white powder, with no odour or taste, which can be used in several food commercialized products. In preferred embodiments, the white powder is also a soluble white powder. Thus, for example, yoghurts supplemented with protein, protein supplements and protein bars are products with a great demand from consumers. In most cases, the products available in the market are supplemented with highly impure whey proteins. Highly pure whey proteins are rather expensive. In the case of allergies or food restrictions these products are no longer a viable option. Products that are supplemented with vegetable proteins (soy and peas) have a characteristic bad taste in addition to the inevitable presence of allergenic proteins and the imbalance in essential amino acids. RuBisCO may be the perfect protein candidate, as it is a non-allergenic protein with a well-balanced proportion of essential amino acids, presenting no taste or odor, and may be easily used to supplement a large variety of food products. But these are just a few of the many examples of protein- enriched food products demanded by consumers. At the moment, there are no products on the market supplemented with vegetable proteins without an associated bad taste and/or the chlorophylls-derived green colour. Those with a more pleasant taste are supplemented with whey proteins. RuBisCO could provide this product line with a tasty new option. RuBisCO may also be applied to other types of foods, from bread and biscuits to pasta, soups, protein puddings, hamburgers, etc. - the options are countless. And all these products would be supplemented with a plant-derived protein rich in essential amino acids ( which from a health perspective may be consider to be even richer than egg protein) whilst being non- allergenic. Further aspects

Embodiments of the invention have provided:

• Methods of obtaining RuBisCO from photosynthetic cells/tissues/organs/entire organisms in a highly pure form as a white powder devoid of taste and odour.

• Methods of obtaining pure RuBisCO as a premium product suitable to be commercialized as a healthy dietary supplement, ingredient or to be directly ingested as a protein nutritional or sports supplement.

• Methods suitable to be scaled-up to an industrial level and produces the pure enzyme at a reasonable price.

• Methods carried out with no toxic compounds (e.g., organic solvents, with the possible exception of ethanol in one embodiment) are employed in the RuBisCO purification process.

• Methods of extraction of the total soluble proteins from photosynthetic cells/tissues/organs/entire organisms with water, containing or not additives;

• Methods of selective precipitation of RuBisCO with sodium phytate and calcium chloride, in the form of a phytate - calcium - RuBisCO insoluble ternary complex;

• Methods of solubilization of RuBisCO, but not of calcium phytate, at high pH values;

• Methods of RuBisCO concentration and/or desalting by nanofiltration, ultrafiltration or microfiltration.

• Methods wherein RuBisCO remains solubilized in water, free from phytate; pigments (e.g., chlorophylls and carotenoids) removal by treatment with activated charcoal, bentonite, etc.; powdery RuBisCO obtained by freeze-drying or spray drying.

• Embodiments comprising extraction of the total soluble proteins from photosynthetic cells/organs/entire organisms with water, containing or not additives.

• Selective precipitation of RuBisCO with ethanol concentrations up to 5 or 10% (v/v).

• Methods of solubilization of RuBisCO. • Methods of RuBisCO concentration and/or desalting by reverse osmosis, nanofiltration, ultrafiltration or microfiltration. Preferably, RuBisCO will remain solubilized in water.

• Methods for the removal of pigments (e.g., chlorophylls and carotenoids) by treatment with activated charcoal, bentonite and the like.

• Methods of obtaining powder by freeze-drying or spray-drying.

• The steps described combining selective RuBisCO precipitation with sodium phytate and calcium chloride with selective RuBisCO precipitation with ethanol, at concentrations up to 5 or 10% (v/v).

Claims

1. A process of obtaining a RuBisCO preparation from photosynthetic material, wherein the process comprises the steps of:

• lysing said photosynthetic material to extract RuBisCO accompanied by a fraction of the chlorophylls of said photosynthetic material;

• performing precipitation with sodium phytate and/or calcium chloride;

• obtaining a phytate-calcium-RuBisCO ternary insoluble complex; and

• performing solubilization of said complex.

2. A process according to claim 1, comprising the further step of obtaining pure RuBisCO dissolved in water.

3. The process according to either claim 1 or claim 2, wherein said concentration of sodium phytate is of 5 to 20 mM; and optionally greater than 10 mM.

4. The process according to any one of the preceding claims, wherein said concentration of calcium chloride is of 5 to 20 mM; and optionally greater than 10 mM.

5. The process according to any one of the preceding claims, wherein said precipitation occurs at a low temperature; optionally said temperature being in a range of 2 to 8 degrees Celsius.

6. The process according to any one of the preceding claims, wherein said solubilization step comprises the use of a glycine buffer or a bicarbonate and/or a carbonate buffer.

7. The process according to any one of the preceding claims, wherein said solubilization step comprises the use of a buffer at a high pH; optionally said pH is greater than 10; optionally said pH is of 11.

8. The process according to any of the preceding claims, wherein said solubilization step comprises the use of one or any combination of any of the following: EDTA (ethylenediaminetetraacetic acid), calcium, calcium chloride, sodium chloride.

9. The process according to any of the preceding claims, further comprising the step of removing phytate by filtration.

10. The process according to claim 8, wherein said removed phytate is recycled for performing one or more further cycles of precipitation.

11. The process according to claim 6 or claim 7, further comprising the step of removing said buffer by filtration.

12. The process according to any one of claims 9 to 11, wherein said step of filtration employs a molecular weight cut-off membrane of 100 kDa or less.

13. The process according to any one of the preceding claims, further comprising a step of precipitation by ethanol with concentrations of up to 15 % (v/v).

14. The process according to claim 13, wherein said concentration of ethanol is of up to 5 or up to 10% (v/v).

15. A process of obtaining a RuBisCO (ribulose-1, 5-bisphosphate carboxylase/oxygenase) preparation from photosynthetic material, wherein the process comprises the steps of: · lysing said photosynthetic material to extract RuBisCO accompanied by a fraction of chlorophylls from said photosynthetic material; and • performing precipitation by ethanol with concentrations of up to 15% (v/v).

16. The process according to claim 15, wherein said ethanol concentrations are up to 5 % (v/v) or up to 10% (v/v).

17. The process according to any of the preceding claims, comprising the steps of employing activated carbon to remove chlorophylls; whereby colourless, odourless and tasteless purified RuBisCO is obtained.

18. The process according to claim 17, wherein said chlorophylls are removed with bentonite.

19. The process according to any of the preceding claims, without any one of the following: addition of enzymatic inhibitors, reducing agents, denaturants, antioxidants, organic solvents (other than ethanol) or removers of phenolic compounds.

20. A RuBisCO preparation obtained by a process according to any one of claims 1 to 19.