WO2009027085A1

WO2009027085A1 - Process for carrying out a chemical reaction in a cell

Info

Publication number: WO2009027085A1
Application number: PCT/EP2008/007050
Authority: WO
Inventors: Donald A. Wellings
Original assignee: Chromatide Ltd
Priority date: 2007-08-29
Filing date: 2008-08-28
Publication date: 2009-03-05
Also published as: GB0716719D0

Abstract

The invention relates to a process for carrying out a chemical reaction in a cell. The process involves providing a first reactant in a cell and either i) initiating self-reaction of the first reactant to produce a product; or ii) contacting the first reactant with a second reactant under conditions such that the first and second reactants optionally with additional reactants react chemically to produce a product. A micro organism may be used as a micron scale chemical reactor. The process is useful in providing polymers, peptides and nucleic acid sequences and in providing a product having a narrow particle size distribution.

Description

PROCESS FOR CARRYING OUT A CHEMICAL REACTION IN A CELL

This invention relates to a process for carrying out a chemical reaction, in particular a chemical reaction at a micro level. The invention more particularly relates to a process for carrying out a chemical reaction within an encapsulate and to the use of a microorganism as a encapsulate or carrier for a reactant for a chemical process and as a reaction zone in which the reaction is carried out. The invention also relates to a reactive substance carried or encapsulated within a biologically produced carrier and to a process for preparing a biologically produced carrier having a reactant therein.

Solution and emulsion phase processes are used in a wide range of process, including synthesis of organic molecules, inorganic particles and organic particles. Synthesis in solution may typically involve multiple-stages with numerous isolation steps to separate intermediates, by-products and the like produced at each stage, before progressing to the subsequent stage in which the intermediates are utilized as feed stocks. These processes may be time-consuming, expensive and may be inefficient as regards yield. The intermediates often require purification to remove excess reagents and reaction byproducts and procedures such as precipitation, filtration, bi-phase solvent extraction, solid phase extraction, crystallization and chromatography may be employed.

Solid support materials are used in a wide range of physical and chemical processes including by way of example synthesis of organic molecules, in particular peptides and oligonucleotides, immobilization of species, support in catalysis and chromatography.

Solid phase synthesis offers some advantages over solution phase synthesis. For example, isolation procedures used in solution phase synthesis may to some extent be avoided by reversibly attaching the target molecule to a solid support. Excess reagents and some of the side-products may be removed by filtration and washing of the solid support. The target molecule may be recovered in high or even essentially quantitative yield in some processes. Recovering high yields in solution phase synthesis is often difficult. In addition, the time required to perform operations on a solid support is typically much less than that required to carry out a comparable stage in a solution phase synthesis. However, the reactivity of substances in solid phase synthesis may be reduced as compared to those observed in solution phase synthesis due to steric hindrance and poor quality solid support used. Provision of a method of synthesizing products which avoids or reduces the disadvantages of both solution and solid phase synthesis would be highly desirable.

We have now found that micro organisms may be used to carry or encapsulate a reactant which may then be reacted with itself or a further reactant so as to produce a reaction product in situ in the encapsulate and avoid or reduce drawbacks associated with solution phase or solid phase synthesis.

Hitherto, encapsulation technology has been concerned with the encapsulation of finished products within polymeric materials and biological carriers provided by micro organisms for example fungi, yeasts, and protozoa are known. The finished product may be for example a dye, a drug, a condiment, a flavour, an aroma, a chemical, a vitamin, an adhesive and the like. Encapsulation is typically employed as a means of controlling the delivery of the encapsulated material to a location at which the product is intended to impart its effect or to protect the encapsulated material from reaction. US-A- 4001480 describes the encapsulation of substances within biological microcapsules with a view to retaining these substances within the micro organism for subsequent release. Encapsulation technology has not been disclosed or suggested for carrying reactant(s) which are to be reacted in the encapsulate.

The invention provides in a first aspect a process for carrying out a chemical reaction comprising providing a first reactant in a cell of a microorganism and either i) initiating self-reaction of the first reactant to produce a product preferably where initiation is provided from outside the microorganism; or ii) contacting the first reactant with a second reactant under conditions such that the first and second reactants react chemically to produce a product.

Suitably, the reactant within the micro organism is not a natural constituent of the micro organism. The second reactant, where present, may be in a cell of the microorganism together with the first reactant or external of the cell such that the cell wall provides a barrier between the first and second reactant. Where the second reactant is in the cell, the first and second reactants suitably do not react together until a further component, for example an initiator, is added to the two reactants and/or reaction conditions are changed such that reaction occurs. Where the second reactant is external of the cell, the second reactant suitably permeates the cell wall alone or in the presence of a solvent to aid transmission of the reactant through the cell wall whereby the reactants contact and react so as to form the product.

Second, third or additional reactants may be included in the cell to provide a multi- component reaction mixture depending on the required reaction or may be present externally of the cell and pass through the cell membrane whereby reaction with the reactant in the cell occurs.

Suitably, reaction of the at least one reactant comprises adding a further reactant, for example an initiator to the microorganism so as to contact the first and optionally second reactant with the so as to initiate reaction upon addition of the further reactant. In a preferred embodiment, the first and optional second reactant comprises monomers and the further reactant comprises a polymerization initiator. The reaction product is suitably a polymer and this may be reacted in the cell with a further reactant to provide a further reaction product.

Any one or more of the reactant(s) may be diluted by blending with a thinner, for example acetone.

Advantageously, in the present process reactions may be performed in solution in the cell so benefiting from advantages of solution phase reaction processes whilst the reactants and product being formed are located within an environment isolated from the environment outside the cell wall and so provide benefits of solid phase technology of reducing the need to separate and purify the products. In traditional solution phase reactions the products are typically isolated at each stage of a multi-step process. This often involves laborious isolation and separation steps such as chromatography, crystallisation, or extractions from immiscible solvents. In the present invention, the reaction chemistry may be manipulated so that the reaction by-products are readily expelled through the cell wall allowing them to be removed by filtration and washing. The present invention provides the advantages of solution phase and solid phase reactions in that on contacting micro organisms carrying the reactants the reaction is essentially carried out in solution within the carrier whilst the reactants and product are isolated within the carrier providing the advantages of solid phase chemistry.

The invention provides the ability to physically isolate reaction components in a convenient form, within the cell. The cells may be treated, for example filtered and washed, prior to eventual release of the product from within the cell.

The cell within which the first reactant is encapsulated is suitably a cell of a micro organism. Suitable micro organisms include yeast for example bakers yeast and brewing yeast, moulds and other fungi and preferably micro organisms having lipid containing cells which suitably provide a lipophilic environment for encapsulating the first reactant. Protozoa may also be employed to encapsulate the first reactant and desirably permit encapsulation of lipid soluble reactants and also other reactants that are permeable through protozoa membranes but that might not be soluble in lipids.

Any micro organism that synthesizes lipids within itself for example yeasts, moulds or other fungi, are suitable for producing the cells into which the reactant(s) to be encapsulated may be absorbed. Examples of suitable fungi include those described in US-A-4001480, EP-A-0085805, GB-A-2424408, WO2005102508 and preferably Saccharomyces cerevisiae (baker's yeast).

The micro organism is preferably in the form of an aqueous slurry but can be used in other continuous phases provided that the continuous phase is immiscible with the solvent inside the cell. Suitably, the reactant(s) are carried in a solvent which is compatible with the cell components and not compatible with the continuous phase of the slurry.

In a further embodiment, the micro organism or cell may be nurtured in a medium that is poor in nitrogen in order to produce excess lipid for example, up to 40 to 60 percent lipid by weight for example as described in US-A-4001480. A higher lipid content may be advantageous for encapsulating lipophilic reactants. The lipid in the cell typically exists as a globule and a higher lipid content provides a greater volume within which the reactant(s) reside in the cell. Advantageously, the cell has a high lipid content although it is not essential for the cell to have a higher lipid content than observed in a natural cell. Suitably the cell has a lipid content of at least 10% and preferably from 40 to 60% by weight of the cell.

Micro organisms having a low lipid content may be treated with a lipid-extending component to extend the lipid content of the micro organism. If one or more of the reactants is insoluble in the lipid in the cell of the micro organism, use of a lipid- extending component in which the reactant is soluble or dispersible may allow the reactant to be encapsulated in the micro organism.

Preferably, the lipid-extending component is a liquid selected from aliphatic alcohols, esters, aromatic hydrocarbons and hydrogenated aromatic hydrocarbons. Examples of especially preferred lipid extending components include primary alcohols having 4 to 15 carbon atoms, iso-butanol, octan-2-ol, tertiary-butanol, diethylene glycol, di-2-ethylhexyl adipate, di-iso-butyl phthalate, butyl benzyl phthalate, acetyl tributyl citrate, 2,2,4- trimethyl-1 ,5-pentanediol iso-butyrate, glyceryl triacetate, glyceryl tributyrate, 2- ethylhexyl acetate, toluene and xylene.

The invention further provides an encapsulation process comprising the steps of: growing a micro organism under conditions that produce a micro organism, preferably having a lipid content of at least 10%, and preferably 40 to 60%, by weight, contacting a reactant with the grown micro organism to pass the reactant into the cell thereby to encapsulate the reactant.

Energy may be applied to the micro organism during contact with the reactant to aid passage of the reactant through the cell wall to encapsulate the reactant, for example heat or pressure may be applied to the micro organism.

The reactant is suitably passively retained within the cell, preferably the lipid in the cell and is not a natural constituent of the micro organism. The reactant within the cell may then be reacted chemically with itself or other another reactant. Desirably, none of the reactants are natural constituents of the cell or the micro organism. Preferably the micro organism is grown within a nutrient medium that is relatively low in nitrogen content and relatively high in carbohydrate content to enhance the formation of lipid within the micro organism.

The cell in which the reactant is encapsulated provides a microcapsule in which the first reactant and second reactant react to produce the product. The cell suitably comprises a fungal cell, bacterial cell or algae. Advantageously, inexpensive micro-organisms for example yeast are employed particularly in larger scale processing due to their low cost. Preferably, the microcapsule comprises fungal cells. Fungal cells are especially preferred as they are commercially available on a large scale and provide economic advantage particularly as compared with traditional solution phase or solid phase chemistry. More preferably, the fungal cell is derived from a yeast. Suitable examples include, Saccharomyces cerevisiae, common bakers yeast, brewing yeast and yeast obtainable as a by-product of ethanol bio-fuel production. The cell of the micro-organism may be alive or dead.

The reactant(s) to be encapsulated and reacted may be lipophilic or may comprise a lipophilic moiety. Preferably, the substance(s) is lipophilic or substantially lipophilic. The term 'substantially lipophilic' as used herein is meant to include those compounds having lipophilic and hydrophilic moieties wherein the lipophilic moiety dominates. The reactant(s) to be encapsulated may also be lipid soluble. The reactant(s) may be derived from a hydrophilic compound which is made lipophilic by chemical modification, such as esterification, etherification, alkylation or alkoylation etc. so as to aid transport of the modified reactant into the cell from the slurry but whilst retaining the required functionality and reactivity to participate in the intended reaction in the cell. Adjustment of physical or chemical parameters, for example the pH of the reactant to be encapsulated may also be used to modify the properties of the reactant, for example to render the reactant more lipophilic.

Where two or more reactants are in the cell, the reaction is suitably initiated by addition of a further component or an alteration in conditions. A preferred example is polymerisation where one monomer for homopolymerisation or two or more monomers are in the cell and the polymerisation reaction is initiated by introduction of an initiator compound. Where one reactant is in the cell, another reactant may be introduced from outside the cell and reaction commences upon contact of the two reactants. In a preferred example, the cell contains an amino acid or a derivative thereof for example glycine trityl ester as a reactant and a second amino acid or derivative thereof, for example a pentafluoroester of Fmoc-alanine, in a lipophilic solvent is added to the continuous phase of an aqueous slurry and passes through the cell wall whereby reaction occurs to form Fmoc-alanine- glycine-trityl ester. This product stays in the cell and may be subsequently removed for example by destruction of the cell or retained in the cell as a reactant for a further process for example addition of further amino acid moieties and the by-product, pentafluorophenol would suitably be expelled from the cell due to its hydrophilic nature. In this way, by-products may be removed from reactions and the desired product obtained in a purer form for recovery or for further reactions.

Suitably, the cell may be employed to separate a desired component from an undesired component, for example a by-product or unreacted reactant. The separation suitably is enabled by the desired and undesired product having different hydrophilicity and the cell or reaction conditions being modified to enable separation.

Suitably the cell wall may be hardened during or after encapsulation of the reactant to reduce the risk of rupture of the cell wall during reaction of the reactants or upon formation of the product. Early rupture may disadvantageously liberate the product or unreacted reactants. Known cell hardening agents may be employed for this purpose for example aldehydes.

The cell wall may be hardened by reaction of the protein in the cell wall with a reactive monomer that can be subsequently polymerised. In a preferred embodiment, acryloyl chloride may be reacted with the cell wall to provide an end-cap for the reactive groups in the cell wall. The free acrylic group of acryloyl chloride capping agent may then be polymerized, thereby toughening the cell wall. The capping agent is suitably selected to impart desired properties upon the cell wall following polymerization to provide functional attributes, for example to aid dispersion of the cell, to impart differing cell membrane transport properties and to impart properties on the cell wall that would allow controlled disruption of the cell wall. Where employed a step for hardening the cell wall may be carried out before or after encapsulation of the reactant.

Once the reactants have reacted and formed the desired product, the product(s) may be released from the cell by disruption of the cell wall or by complete destruction of the cell. This may be carried out by biochemical means for instance by the use of an enzymatic process, for example by contact with a proteolytic enzyme, or by a chemical process for instance by the use of concentrated acid such as concentrated hydrochloric acid or alkali. The disruption can also be done mechanically, for example by ultrasonics. However, the release mechanism is not limited to these processes.

The invention further provides a process by which a product of a pre-determined particle size may be produced. In a further aspect, the invention provides a process for the production of a product having a pre-determined particle size comprising providing a first reactant in a cell having an internal diameter the same or larger than the pre-determined particle size, introducing a second reactant into the cell and contacting the first reactant with a second reactant under conditions such that the first and second reactants react chemically to produce a product and wherein the first and second reactants are introduced in such quantity as to produce a volume of product not greater than the internal volume of the cell.

Preferably the micro organism has a cell size from 1 to 20 microns diameter and the product is suitably produced in a particle size from 1 to 20 microns.

The invention provides in a further aspect a product produced within a cell and having a particle size of 1 to 20 microns and a particle size range of less than 1 micron up to the maximum particle size feasible within a particular cell type. For example, baker's yeast has a cell size of 6 micron so it is possible to prepare particles up to 6 micron in this cell.

The reaction produces a product that is constrained by the size of the cell in which the product is made. The reactant(s) reside in the lipid globule present in the cell and the particle size of the product will be at least the size of the lipid globule thereby providing a means of controlling the lower limit of the particle size. By including a lipid extending component, the lipid globule may be increased in size to almost the size of the cell so providing a narrow range between the smaller lipid globule size and the larger internal cell volume and thereby constraining the particle size of the product within narrow limits. This control over particle size is especially beneficial in the formation of polymers as the need to classify and sieve product particles as is typically required in for instance emulsion polymerisation may be avoided.

Advantageously, the present invention provides a way to physically contain chemical reaction processes and is useful in a range of applications, particularly in simplifying repetitive processes including peptide synthesis, oligonucleotide synthesis and oligosaccharide synthesis.

In a further aspect, the invention provides the use of a micro organism as a micron-scale chemical reactor to perform a chemical reaction.

The invention also provides the use of a micro organism containing an introduced reactant in a cell as a chemical reactor in which the reactant is reacted with a second reactant introduced to the cell whereby to produce a product and optionally releasing the product from the cell.

The invention further provides a reaction product, preferably a polymer, contained in a cell and wherein the product has been produced in the cell.

In an especially preferred embodiment, the cell employed in the present invention is a yeast cell.

Known solid phase supports generally comprise polymer particles of a particular size and physical nature to suit the application. For ease of use these polymer particles are often spherical and have a defined particle size distribution. The spherical nature of the particles improves the flow and filtration characteristics of the polymer. Although the uses of solid supports have operational advantages there are disadvantages to the solid phase approach. For example, commercially available supports commonly used for solid phase synthesis of peptides and oligonucleotides may be expensive. Polymeric particles may typically be made by a dispersion or emulsion polymerization process in which a solution of monomers is dispersed in an immiscible solvent (continuous phase) prior to initiation of the polymerization. The polymer particles formed are typically then filtered, washed and classified. These processes are disadvantageous in some respects including monomer loss to the continuous phase, generation of a range of particle sizes and generation of fine particles during the polymerization. Loss of monomers to the continuous phase may be inefficient in terms of both raw material and environmental costs. Classification of the polymer particles to isolate the particle size required for a particular application may be a laborious and complex process, typically involving sieving and (or) air classification which may lead to losses in yield. 'Fines' particles are usually produced. These fines may be problematic in isolation of the polymer beads and may require additional processing, for example settling and decantation for their removal.

In addition to undesirable costs of manufacture and wastage during preparation certain disadvantages may arise with the physical properties of the known polymeric particles. Microporous polymers and macroporous polymers are generally used and their manufacture may be expensive and complex. Microporous polymers have a relatively low level of cross-linker which allows the polymer particles to solvate and consequently swell in suitable solvents.

Macroporous polymers have a high level of cross-linker in the polymer matrix and contain large pores. These polymer particles are generally rigid and have good flow characteristics in packed columns.

Problems associated with the costs of production, wastage, physical integrity of the support and poor product performance may be ameliorated by providing a simple process for the manufacture of polymers as provided by the present invention.

In a further aspect, the invention provides a process for the production of a polymer comprising providing a cell containing a monomer, initiating polymerization of the monomer in the cell to produce the polymer.

In a preferred embodiment, the polymer produced is a copolymer and the process comprises the step of introducing a second monomer into the cell, reacting the two or more monomers to produce a copolymer of the two or more monomers. The reaction between the two or more monomers may occur upon contact or require initiation depending on the particular monomers.

The present invention may be applied in the production of organic polymers and inorganic for example organic polymers generated from encapsulated divinylbenzene monomer and styrene monomer, and inorganic polymers, for example silica generated by polymerization of encapsulated alkoxysilanes.

Controlling the particle size of the product also provides advantage in producing polymers. For example, monomers for the polymer backbone and cross linking monomers of the intended particles may be encapsulated optionally with the other components, for example porogens and co-solvents if required. The mixture comprising the reactants and optional further components may then be polymerized by initiation of the polymerization reaction employing polymerization initiation means known to those skilled in the art. The natural lipids contained within the cells may also suitably act as porogens for the polymer particles.

The invention provides a comparatively inexpensive and facile process for generation of micron size polymeric particles. Advantageously, polymers produced according to this invention may be employed in a wide range of applications including by way of example, the manufacture of ink toners, polytetrafluoroethylene (PTFE), paints, catalysts including transition metal, chemocatalysts and biocatalysts, solid phase synthesis supports and stationary phases for chromatography.

Polymeric particles producible according to the present invention are especially useful in chromatography applications as stationary phases. In certain modes of chromatography the cost of stationary phases may be high and restrict usage. The application of stationary phases in chromatographic separations is very extensive including for example complex high-technology separations used in the pharmaceutical and biotechnology industry for the purification of high value products using preparative chromatography and large-scale separations employed in the mining industry. A large portion of the world's Palladium, a critical component in catalytic converters and other industrial processes, may be refined using immobilized crown ethers (Traczyk, F. P.; Bruening, R. L.; Izatt, N. E. "The Application of Molecular Recognition Technology (MRT) for Removal and Recovery of Metal Ions from Aqueous Solutions"; In Fortschritte in der Hydrometallurgie; 1998, Vortrage beim 34. Metallurgischen Seminar des Fachausschusses fuer Metallurgische Aus-und Weiterbildung der GDMB; 18-20 November 1998; Goslar).

Polymeric particles produced according to the present invention may also be employed in solid phase extraction and in the preparation of solid phase reagents is also known in the chemical, pharmaceutical and biotechnology industry.

Polymer particles prepared according to the invention are particularly useful in supporting precious metal catalysts, for example palladium catalysts. A particular advantageous example is palladium acetate. In a preferred embodiment, palladium acetate is supported on a polyurea or a polyacrylamide polymer produced according to the present invention.

The polymer particles prepared according to the invention are particularly useful for as stationary phases for applications in many modes of chromatography including ion exchange, reversed phase, normal phase, chiral, affinity and size exclusion chromatography. The particles prepared according to the invention are especially useful in affinity chromatography, for example in the immobilisation of affinity ligands for example protein A. Affinity chromatography is used predominantly for the separation of biological products for example biopharmaceuticals. The affinity ligand is suitably immobilized on a stationary phase. This ligand has a particular affinity for a component of a biological mixture to be contacted with the support. The affinity may be based on any form of interaction for example a specific biological interaction such as seen with an enzyme and substrate, a receptor and ligand and an antigen and antibody.

In a preferred embodiment the affinity ligand comprises Protein A and is used to interact with immunoglobulins. Protein A binds to the Fc region of several immunoglobulin antibodies and many biopharmaceuticals are based on immunoglobulins.

The particles prepared according to the invention may be used to immobilize species for example antibodies, oligonucleotides, enzymes or fluors and may be useful in diagnostic assays. The invention is illustrated by the following non-limiting examples.

Example 1 - Preparation of Co-polystyrene-divinylbenzene Beads

Yeast (Saccharomyces cerevisiae, 6Og) was slurried in water (400cm³). The mixture was stirred rapidly and heated to 5O⁰C.

An emulsion was prepared containing styrene (4g), divinylbenzene (16g), toluene (4g), Span 80 (0.5g) and water (20cm³). This emulsion was added drop-wise to the stirred yeast slurry prepared above. The whole mixture was then stirred at 5O⁰C for 2h to encapsulate the monomers and toluene within the yeast cells.

Polymerization was initiated by addition of a solution of ammonium persulfate (4g) in water (20cm³). The temperature was increased to 7O⁰C and the mixture stirred for 2h.

The encapsulated copolystyrene-divinylbenzene particles were recovered by enzymatically destroying the yeast. Household biological detergent (2g) was added to the slurry and the mixture stirred at 6O⁰C and for 2h.

The polymer particles were recovered by filtration and washing with water (3x100cm³), methanol (3x100cm³) and acetone (3x100cm³) before air drying to yield (11.8g) of copolystyrene-divinylbenzene particles. The particles, observed under a microscope, had a diameter of ~4 micron when compared to a 10 micron chromatographic stationary phase.

Example 2 - Preparation of vinylsilica particles

Yeast (Saccharomyces cerevisiae, 28g) and triethoxyvinylsilane (15g) were mixed together to form a paste. The paste was slurried in water (150cm³), stirred rapidly and heated to 5O⁰C.

The temperature was maintained at 50⁰C for 2h during which the encapsulated triethoxyvinylsilane would start to polymerize. Concentrated hydrochloric acid (20cm³, 37%w/v) was added to catalyze the formation of vinylsilica particles and the mixture stirred for 2h.

The encapsulated vinylsilica particles were recovered by filtration and washing with water (3x100cm³) and methanol (6x100cm³) before air drying. The encapsulated product was heated in an oven at 11O⁰C for 6Oh.

The yeast was then destroyed by boiling the encapsulated vinylsilica particles in concentrated hydrochloric acid (20cm³, 37%w/v) for 2h.

The vinylsilica particles were recovered by filtration and washing with water (3x50cm³), methanol (3x50cm³) and acetone (6x50cm³) before air drying to yield (7.4g) of vinylsilica particles. The particles, observed under a microscope, had a diameter of ~5 micron when compared to a 10 micron chromatographic stationary phase.

Example 3 - Preparation of encapsulated Co-polvstyrene-chloromethylstyrene- divinylbenzene Beads

An emulsion was prepared containing styrene (4g), divinylbenzene (16g), 4- chloromethylstyrene (0.2g), toluene (4g) and water (20cm³). This emulsion was added drop-wise to the stirred yeast slurry prepared above. The whole mixture was then stirred at 5O⁰C for 2h to encapsulate the monomers and toluene within the yeast cells.

The encapsulated copolystyrene-chloromethylstyrene-divinylbenzene particles were recovered by filtration and washing with water (3x100cm³), methanol (3x100cm³) and diethyl ether (3x100cm³) before air drying (yield 34.8g). Example 4 - Reaction of encapsulated copolystyrene-chloromethylstyrene- divinylbenzene particles with ethylenediamine

Encapsulated copolystyrene-chloromethylstyrene-divinylbenzene particles (34.3g) were suspended in 1 ,2-ethylenediamine at room temperature and left for 16h.

The particles were recovered by filtration and washing with water (3x100cm³), methanol (3x100cm³) and diethyl ether (3x100cm³) before air drying (yield 17g).

Any remaining yeast was then destroyed by boiling the particles (15g) in concentrated hydrochloric acid (500cm³, 37%w/v) for 5h.

The amino functional polymer particles produced in this example were recovered by filtration and washing with water (3x100cm³), methanol (3x100cm³) and diethyl ether (3x100cm³) before air drying (yield 6.9g).

The particles, observed under a microscope, had a diameter of ~4 micron when compared to a 10 micron chromatographic stationary phase. The material produced gave a positive Ninhydrin assay confirming the presence of amino functional groups on the particles.

Claims

1. A process for carrying out a chemical reaction comprising providing a first reactant in a cell in a microorganism and either i) initiating self-reaction of the first reactant to produce a product where initiation is provided from outside the microorganism; or ii) contacting the first reactant with a second reactant under conditions such that the first and second reactants react chemically to produce a product.

2. A process according to claim 1 comprising the steps of: growing a micro organism under conditions that produce a micro organism, contacting a first reactant and optionally a second reactant with the grown micro organism to pass the at least one reactant into a cell in the microorganism thereby to encapsulate the at least one reactant prior to step i) or step ii) as defined in claim 1.

3. A process according to claim 1 or claim 2 wherein the cell has a lipid content of at least 10% by weight of the cell.

4. A process according to any one of the preceding claims comprising adding an initiator to the microorganism so as to contact the first and optionally second reactant with the initiator and initiate reaction upon addition of the initiator.

5. A process for the production of a product having a pre-determined particle size comprising providing a first reactant in a cell having an internal diameter the same or larger than the pre-determined particle size, introducing a second reactant into the cell and contacting the first reactant with a second reactant under conditions such that the first and second reactants react chemically to produce a product and wherein the first and second reactants are introduced in such quantity as to produce a volume of product not greater than the internal volume of the cell.

6. A process according to claim 5 wherein the first reactant comprises a polymer which has been produced in the cell.

7. A process according to any one of the preceding claims comprising a second reactant which is either: i) in the cell of the microorganism together with the first reactant and reaction occurs on addition of a third component or on alteration of the reaction conditions so as to initiate reaction; or ii) external of the cell such that the cell wall provides a barrier between the first and second reactant and the second reactant permeates the cell wall alone or in the presence of a solvent to aid transmission of the reactant through the cell wall whereby the reactants contact and react so as to form the product.

8. A process according to any one of the preceding claims wherein the micro organism is selected from a yeast, a mould, a fungi and protozoa.

9. A process according to any one of the preceding claims in which the microorganism is treated with a lipid-extending component.

10 Use of a micro organism as a micron-scale chemical reactor to perform a chemical reaction to produce a product from reactants which are not present in the micro organism.

11. Use according to claim 10 of a micro organism containing an introduced reactant in a cell of the micro organism as a chemical reactor in which the reactant is reacted with a second reactant introduced to the cell whereby to produce a product and optionally releasing the product from the cell.

12. Use according to claim 11 or claim 12 wherein the product is released from the cell by disruption of the cell wall or destruction of the cell.

13. A product produced within a cell according to a process as defined in any one of claims 1 to 9 and having a particle size of 1 to 20 microns.

14. A product according to claim 13 having a particle size range of less than 1 micron up to the maximum internal dimensions of the cell.

15. A product according to claim13 or claim 14 comprising a polymer or a peptide.

16. Use of a product comprising a polymer according to any one of claims 13 to 15 as a chromatography stationary phase.

17. A process for the production of a polymer comprising providing a cell containing a monomer and optionally a comonomer, initiating polymerization of the monomer in the cell to produce the polymer and optionally further reacting the polymer in the cell.