Functionalised biopolymer particle preparation

FIELD

[0001] The present disclosure relates generally to methods for preparing biopolymer particles, and more particularly, to methods for preparing functionalised biopolymer particles. The functionalised biopolymer particles are suitable for, but not limited to, the immobilisation of enzymes for use in applications requiring enzyme catalysis while having improved environmental characteristics. The present disclosure also provides functionalised biopolymer particles obtained by the inventive methods and uses thereof.

BACKGROUND

[0002] Biopolymers are an important development in the reduction of the environmental footprint of consumer products. Biopolymers and biopolymer particles are often biodegradable as well as being derived from renewable and sustainable raw materials.

[0003] In industrial processes, synthetic polymers are often used as solid supports. For example, in the form of beads upon which catalysts may be immobilised. For catalytic transformations, heterogeneous catalysts are preferred as they can be readily separated and reused on a large scale. They are also compatible with many industrial process configurations. Here, materials made from polymers are widely used because of their adaptability, durability and price.

[0004] While industrial processes have generally employed synthetic catalysts such as transition metal coordination complexes, the use of biological catalysts, e.g. enzymes, has grown. Biocatalysis may be advantageous as it can offer stereospecific transformations, milder reaction conditions, and may have a preferable safety and environmental profile.

[0005] However, the enzymes used for such processes are typically immobilised on a polymer substrate such as acrylic or polystyrene beads. The bead form factor facilitates mixing during reaction and easy separation from the end products. Synthetic polymers such as acrylic or polystyrene are mainly derived from petroleum and gas as raw materials, meaning that they are incompatible with the environment and reliant on an unsustainable resource. Polymer particles in particular, pose serious ecological problems because they often remain in ecosystems following disposal of the product. Moreover, when biocatalysis is applied in the food industry, for example for the conversion of corn syrup into high fructose corn syrup, there are additional challenges because it is desirable that all traces of solvents and unplasticised monomers arising from the manufacturing process of the polymer support, which may be hazardous to human health, are removed before use in the processing of foodstuffs.

[0006] Biopolymer particles may therefore be an attractive alternative to synthetic polymer supports in industrial processes such as biocatalysis to realise further environmental and safety benefits. To be commercially viable, a high degree of immobilisation of the catalyst such as an enzyme should be achieved. Moreover, as enzymes are generally costly reagents, it will usually be desirable for the immobilised enzyme and support to be amenable to multiple reuse cycles while retaining a high degree of catalytic activity.

[0007] Biopolymer particles prior to the present disclosure may not meet some or all of these requirements.

[0008] Thus, there is a need in the art for methods of preparing biopolymer particles suitable for use in industrial processes. There is a need, for example, for a generally applicable approach that facilitates the immobilisation of catalysts such as enzymes, wherein the biopolymer-immobilised catalysts exhibit a high degree of activity that is preserved during multiple washing and re-use cycles. The methods of the present disclosure seek to address this unmet need.

SUMMARY

[0009] In one aspect, the present disclosure provides a method for preparing functionalised biopolymer particles, said method comprising the steps of: (i) oxidising the biopolymer particles with an agent to form oxidised biopolymer particles, wherein the degree of oxidation of the oxidised biopolymer particles is less than or equal to about 25%; and (ii) reacting said oxidised biopolymer particles with a compound comprising one or more hydrophobic moieties.

[0010] In various embodiments, the biopolymer is a polysaccharide, preferably cellulose. The cellulose may be selected from the group consisting of virgin, recycled, pulp, and microcrystalline cellulose and combinations thereof.

[0011] In various embodiments, the biopolymer particles are in the form of a hydrogel for step (i) of the method of the first aspect.

[0012] In various embodiments, the biopolymer particles are prepared by extruding a dispersed phase into an anti-solvent to form particles of the biopolymer, wherein the dispersed phase comprises the biopolymer in a solvent, and wherein each of the solvent and anti-solvent comprises water. In some embodiments, extruding the dispersed phase into an anti-solvent to form particles of the biopolymer comprises extruding the dispersed phase through a fluid medium into a mould and then contacting the extruded dispersed phase with the anti-solvent.

[0013] In various embodiments, the biopolymer particles are prepared by a method comprising (a) a membrane emulsification of a dispersed phase into a continuous phase wherein the dispersed phase comprises the biopolymer in a solvent, and wherein passing the dispersed phase through the membrane forms an emulsion of the biopolymer in the continuous phase; and (b) a phase inversion with an anti-solvent to form particles of the biopolymer; wherein each of the solvent and anti-solvent comprises water.

[0014] In various embodiments, the solvent of the dispersed phase further comprises an ionic liquid.

[0015] In various embodiments, the anti-solvent is substantially free of organic solvents. The anti-solvent may further comprise an ionic liquid. In further embodiments, the anti-solvent consists of water.

[0016] In various embodiments, step (i) of the method of the first aspect is conducted at room temperature. The oxidising agent may be selected from the group consisting of periodic acid and salts thereof.

[0017] The compound comprising one or more hydrophobic moieties may be a hydrocarbyl amine, preferably a primary hydrocarbyl amine. Step (ii) of the method of the first aspect may further be conducted at room temperature.

[0018] In various embodiments, the method of the first aspect further comprises the step of

(iii) reducing the biopolymer particles of step (ii) with a reducing agent.

[0019] In various embodiments, the hydrocarbyl amine is a hydrocarbyl diamine, preferably wherein each amine group of the diamine is a primary amine. The hydrocarbyl diamine may be selected from the group consisting of C6-C14 hydrocarbyl diamines, preferably from the group consisting of Ce-C hydrocarbyl diamines.

[0020] In various embodiments, the method of the first aspect further comprises the step of

(iv) contacting the reduced biopolymer particles with an enzyme to form enzyme functionalised biopolymer particles. The enzyme may be a lipase, preferably selected from the group consisting of Candida antartica lipase B, Thermomyces lanuginosus lipase, and Pseudomonas cepacia lipase. In various embodiments, the enzyme is immobilised on the biopolymer particle. [0021] In a further aspect, the present disclosure provides functionalised biopolymer particles obtained by the methods described herein. Features described herein in the context of the methods are also therefore applicable to the functionalised biopolymer particles obtained by the methods.

[0022] In various embodiments, the functionalised biopolymer particles are approximately spherical. In various embodiments the functionalised biopolymer particles have a diameter from about 0.2 mm to about 3 mm.

[0023] In a further aspect, the present disclosure provides for the use of the functionalised biopolymer particles obtained by the methods described herein for immobilising an enzyme. Features described herein in the context of the methods and particles are applicable to the use.

[0024] In yet another aspect, the present disclosure provides a method for performing an enzyme-catalysed reaction wherein the enzyme catalysing the reaction is immobilised on functionalised biopolymer particles prepared by the methods described herein. Features described herein in the context of the preparation method and obtained particles are applicable to this method.

[0025] In a still further aspect, the present disclosure provides for the use of enzyme immobilised biopolymer particles prepared by the methods described herein for catalysing a reaction. Features described herein in the context of the preparation method and obtained particles are applicable to this use.

[0026] These aspects and embodiments are set out in the appended independent and dependent claims. It will be appreciated that features of the dependent claims may be combined with each other and with features of the independent claims in combinations other than those explicitly set out in the claims. Furthermore, the approaches described herein are not restricted to specific embodiments such as those set out below, but include and contemplate any combinations of features presented herein.

[0027] The foregoing and other objects, features, and advantages of the present disclosure will appear more fully hereinafter from a consideration of the detailed description that follows along with the accompanying drawings. It is to be expressly understood, however, that the drawings are for illustrative purposes and are not to be construed as defining the limits of the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a schematic representation of an embodiment wherein the biopolymer particles are prepared by extruding a dispersed phase into an anti-solvent.

[0029] Figure 2 contains a schematic representation of an embodiment wherein the biopolymer particles are prepared by membrane emulsification (Figure 2(a)), and a representation of a further embodiment wherein the emulsion is cooled as described herein (Figure 2(b)).

[0030] Figure 3 shows the degree of enzyme adsorption on unmodified cellulose (CNT), dialdehyde-cellulose (DAC10), 1 ,6-diaminohexane aminated (1 ,6 DABs), 1 ,8-diaminooctane aminated (1 ,8 DABs), and 1 ,10-diaminodecane aminated (1 ,10 DABs) beads for each of Candida antartica lipase B (CaLB), Thermomyces lanuginosus lipase (TL Lipolase), and Pseudomonas cepacia lipase (AMANO PS). The degree of adsorption is determined by measuring the percentage enzyme adsorbed from solution and the mass fraction of enzyme loading after incubation with the beads. The unmodified beads are indicated by the ‘CNT’ bar. Error bars represent standard deviations (n = 3).

[0031] Figure 4 shows the initial lipase activity as determined according to the Examples herein and expressed as units per gram (ll/g) for unmodified beads prepared using ethanol or water as the anti-solvent according to the extrusion method described herein onto which Candida antartica lipase B (CaLB), Thermomyces lanuginosus lipase (TL Lipolase), or Pseudomonas cepacia lipase (AMANO PS) was subsequently adsorbed. Error bars represent standard deviations (n = 3)

[0032] Figure 5 shows initial (Cycle 1) lipase activity as determined according to the Examples herein and expressed as Units per gram (dry weight of beads) for (a) Candida antartica lipase B (CalB), (b) Thermomyces lanuginosus lipase (TL Lipolase), and (c) Pseudomonas cepacia lipase (Amano PS), each of which are immobilised on aminated beads functionalised with 1 ,6- diaminohexane (1 ,6-DAB), 1 ,8-diaminooctane (1 ,8-DAB), or 1 ,10-diaminodecane (1 ,10-DAB). Also shown are the lipase activities of the same after repeated cycles of recycling, wherein in each recycling step the beads were washed with PBS to remove substrate and products from the preceding activity measurement. Error bars represent standard deviations (n = 3)

[0033] Figure 6 shows lipase activity as determined according to the Examples herein and expressed as Units per gram (dry weight of beads) for (a) Candida antartica lipase B (CalB), (b) Thermomyces lanuginosus lipase (TL Lipolase), and (c) Pseudomonas cepacia lipase (Amano PS), each of which are immobilised on aminated beads functionalised with 1 ,6- diaminohexane (1 ,6-DABs), 1 ,8-diaminooctane (1 ,8-DABs), or 1 ,10-diaminodecane (1 ,10- DABs). The amount of lipase activity remaining after 1 , 2, 3, and 4 months of storage at 4°C in deionised water is shown in each case. Error bars represent standard deviations (n = 3)

[0034] Figure 7 is a scanning electron micrograph image of enzyme immobilised, functionalised biopolymer particles prepared from cellulose and (i) 1 ,6-diaminohexane, (ii) 1 ,8- diaminooctane, or (iii) 1 ,10-diaminodecane as described in the Examples herein. Representative images of whole beads, the bead surface, and the internal structure of said beads are shown in each case.

DETAILED DESCRIPTION

[0035] While various exemplary embodiments are described or suggested herein, other exemplary embodiments utilizing a variety of methods and materials similar or equivalent to those described or suggested herein are encompassed by the general inventive concepts. Those aspects and features of embodiments which are implemented conventionally may not be discussed or described in detail in the interests of brevity. It will thus be appreciated that aspects and features of apparatus and methods described herein which are not described in detail may be implemented in accordance with any conventional techniques for implementing such aspects and features.

[0036] As used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0037] In this specification, unless otherwise stated, the term "about" modifying the quantity of a component refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making concentrates, mixtures or solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the materials employed, or to carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term "about", the claims include equivalents to the quantities.

[0038] The ranges provided herein provide exemplary amounts of each of the components. Each of these ranges may be taken alone or combined with one or more other component ranges. [0039] As used herein, the term “at least” includes the end value of the range that is specified. For example, “at least 10 cm” includes the value 10 cm.

[0040] As used herein, wt% means “weight percentage” as the basis for calculating a percentage. Unless indicated otherwise, all % values are calculated on a weight basis, and are provided with reference to the total weight of the product in which the substance is present. For example, % water in the solvent of the dispersed phase refers to the wt% water based on the total weight of the solvent. Similarly, % biopolymer in the dispersed phase refers to wt% biopolymer based on the total weight of the dispersed phase.

[0041] As used herein, “substantially free” means no more than trace amounts, i.e. the amount of the substance(s) concerned is negligible. In various embodiments, “substantially free” means no more than 1000 ppm, preferably no more than 100 ppm, more preferably no more than 10 ppm, even more preferably no more than 1 ppm of the substance(s) concerned.

[0042] In all aspects of the present disclosure, the disclosure includes, where appropriate, all enantiomers and tautomers of the compounds disclosed herein. A person skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.

[0043] Some of the compounds disclosed herein may exist as stereoisomers and/or geometric isomers - e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present disclosure contemplates the use of all the individual stereoisomers and geometric isomers of those compounds, and mixtures thereof. The terms used in the claims encompass these forms.

[0044] The general inventive concept is centred on providing a method for preparing functionalised biopolymer particles, where the functionalised biopolymer particles are suitable for use in applications in, but not limited to, biocatalysis, food, biomedicine and pharmaceuticals and have improved environmental benefits. In particular, the biopolymer particles of the present disclosure are functionalised by reaction with compounds comprising hydrophobic moieties, which alters the properties of the biopolymers in order to enable or improve their suitability for use in one or more of the above-mentioned applications.

[0045] Thus, in a first aspect, the present disclosure provides a method for preparing functionalised biopolymer particles wherein the biopolymer particles are oxidised with an agent to form oxidised biopolymer particles. The oxidation process modifies the nature of the functional groups present in and/or on the biopolymer particles, permitting subsequent functionalisation of the oxidised biopolymer particles. Thus, in the first aspect, the oxidised biopolymer particles are reacted with a compound comprising one or more hydrophobic moieties.

[0046] In the present disclosure, it has been found that the hydrophobic functionalisation of the biopolymer particles according to the present disclosure confers advantageous physicochemical properties over unmodified biopolymer particles. In particular, hydrophobic functionalisation of biopolymer particles according to the methods disclosed herein has been found to enhance the degree of adsorption of enzymes by the particles. Moreover, enzymes adsorbed in this manner have been found to be more resistant to being washed off during the washing and recycling of the biopolymer particles, leading to enhanced retention of enzymatic activity during multiple re-use cycles. Similarly, the functionalised biopolymer particles of the present disclosure upon which enzymes have been immobilised exhibit good retention of enzymatic activity during longer periods of storage. Moreover, the functionalised biopolymer particles exhibit good mechanical stability during preparation and their subsequent use, including during storage and recycling.

[0047] For ease of reference, these and further features of the present disclosure are now discussed under appropriate section headings. However, the teachings under each section are not limited to the section in which they are found. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

Biopolymer particles

[0048] All aspects of the present disclosure concern biopolymer particles. By the term “biopolymer” is meant a polymer produced by living organisms. In other words, a polymeric biomolecule. There are three main classes of biopolymers, classified according to the monomeric units used and the structure of the biopolymer formed: polynucleotides (RNA and DNA), which are polymers composed of 13 or more nucleotide monomers; polypeptides, which are polymers of amino acids; and polysaccharides, which are typically polymeric carbohydrate structures. Other examples of biopolymers include rubber, suberin, melanin, chitin and lignin.

[0049] In various embodiments of the present disclosure, the biopolymer is selected from the group consisting of polynucleotides, polypeptides and polysaccharides. Preferably, the biopolymer is selected from the group consisting of polypeptides and polysaccharides. More preferably, the biopolymer is a polysaccharide, for example, starch, cellulose, chitin, chitosan or glycogen. Even more preferably the biopolymer is starch or cellulose. Most preferably the biopolymer is cellulose.

[0050] Cellulose is a linear polymer made up of p-D-glucopyranose units covalently linked with 1^4 glycosidic bonds. Cellulose may be obtained from many different sources and the present disclosure is not necessarily limited as to the origin, form, or other characteristics of the cellulose. Cellulose is typically obtained from plant sources, for example from virgin or recycled wood pulp. Pulp is a lignocellulosic fibrous material prepared by chemically or mechanically separating cellulose fibers from wood, fiber crops, waste paper, or rags. Cellulose may be obtained from virgin sources or advantageously from recycled sources.

[0051] In some embodiments, the biopolymer is selected from the group consisting of virgin, recycled, pulp, and microcrystalline cellulose, and combinations thereof. In some embodiments, the biopolymer is virgin cellulose. In some embodiments, the biopolymer is recycled cellulose. In some embodiments, the biopolymer is pulp cellulose. Preferably, the biopolymer is microcrystalline cellulose.

[0052] Microcrystalline cellulose (MCC) is typically made from high-grade, purified wood or cotton cellulose. Hydrolysis is used to remove amorphous cellulose until the microcrystalline form remains. With its amorphous cellulose portions removed, it becomes an inert, white, free- flowing powder. It can be processed in a number of ways, for example through reactive extrusion, steam explosion, and acid hydrolysis. An example of a commercially available MCC is Avicel® produced by DuPont.

[0053] The term “particle” is used interchangeably herein with “bead” and refers to a discrete solid entity with defined size and shape. The size of the biopolymer particles of the present disclosure is not limited, and the skilled person will be able to select sizes according to a desired application. The size of the particles may be readily identified by a person skilled in the art, for example, using an optical microscope image and image analysis software with a suitable detection algorithm (e.g. Imaged using an edge detection algorithm), laser diffraction with commercially available equipment such as Mastersizer from Malvern Panalytical (e.g. Mastersizer 3000), with an appropriately sized sieve, or by using a caliper.

[0054] In various embodiments of the present disclosure, the biopolymer particles are approximately spherical. Approximately spherical biopolymer particles may be advantageous in certain applications, for example in biocatalysis, where such a shape can facilitate industrial processing and recovery processes such as filtration.

[0055] As recited herein, “diameter” takes its usual meaning and is used in relation to approximately spherical biopolymer particles. Thus, the skilled person will understand that the diameter of an approximately spherical particle as recited herein will be approximately the same when measured in any direction through the centre of said particle. In some embodiments, the particles of beads may have a diameter of at least about 1 pm. In some embodiments, the particles of beads may have a diameter of at least about 10 pm. In some embodiments, the particles of beads may have a diameter of at least about 25 pm. In some embodiments, the particles of beads may have a diameter of at least about 50 pm. In some embodiments, the particles of beads may have a diameter of at least about 100 pm.

[0056] In some embodiments, the particles of beads may have a diameter of less than about 5 mm. In some embodiments, the particles of beads may have a diameter of less than about

4 mm. In some embodiments, the particles of beads may have a diameter of less than about

3 mm. In some embodiments, the particles of beads may have a diameter of less than about

2 mm. In some embodiments, the particles of beads may have a diameter of less than about

1 mm.

[0057] In various embodiments, the functionalised biopolymer particles have a diameter of from about 1 pm to about 3 mm. In various embodiments, the functionalised biopolymer particles have a diameter of from about 10 pm to about 3 mm. In various embodiments, the functionalised biopolymer particles have a diameter of from about 25 pm to about 3 mm. In various embodiments, the functionalised biopolymer particles have a diameter of from about 50 pm to about 3 mm.

[0058] In various embodiments, the functionalised biopolymer particles have a diameter of from about 0.1 mm to about 3 mm. In various embodiments, the functionalised biopolymer particles have a diameter of from about 0.15 mm to about 3 mm. In various embodiments, the functionalised biopolymer particles have a diameter of about 0.20 mm to about 3 mm.

[0059] In various embodiments, larger particles may be preferred to facilitate their use in industrial processes, for example for ease of separation. Thus, in various embodiments, the particles or beads may have a diameter greater than or equal to about 0.2 mm. In various embodiments, the particles or beads may have a diameter of from about 0.2 mm to about 3 mm, from about 1 to about 3 mm or from about 1 to about 2 mm. [0060] Biopolymer particles may be obtained from various methods of production in a form wherein said particles are wetted or immersed in a solvent such as water. Such particles may be referred to as “wet” beads and may be provided in this form for further use. Alternatively, particles may be subsequently dried to provide “dry beads”. In the present disclosure, it is preferable that the biopolymer particles oxidised in the method of the first aspect have not previously been dried.

[0061] In various embodiments of the disclosure, the biopolymer particles are in the form of a hydrogel for step (i) of the method of the first aspect. A hydrogel is defined by IIIPAC as a gel in which the swelling agent is water. As used herein, the term “hydrogel” means a non-fluid polymer network (i.e. formed by the biopolymer) that is expanded throughout its whole volume by a fluid, namely water.

[0062] Biopolymer particles in the form of a hydrogel may be preferred as they can provide a higher effective surface area for reaction in the oxidation step (i) than biopolymer particles that are not in the form of a hydrogel, for example solid particles. An increased effective surface area in turn leads to an increased density of oxidised functional groups for subsequent functionalisation compared to non-hydrogel biopolymer particles. In particular, the enhanced effective surface area of the hydrophobically functionalised biopolymer particles may lead to an enhanced degree of adsorption of enzyme on said particles. Thus, an enhanced degree of enzymatic activity per particle may be achieved with biopolymer particles in the form of a hydrogel compared to non-hydrogel particles such as solid particles. When considering the commercial viability of industrial applications, it is preferable to maximise the activity per particle for reasons of efficiency and cost-effectiveness.

Oxidation

[0063] The oxidation step (i) in the method of the first aspect comprises oxidising the biopolymer particles with an agent to form oxidised biopolymer particles. This agent can be an oxidising agent and the skilled person will be able to select suitable agents capable of oxidising biopolymer particles. The present disclosure is not limited in this respect.

[0064] Non-limiting examples of agents capable of oxidising biopolymer particles are halogens such as chlorine, hydrogen peroxide, peracetic acid, chlorine dioxide, nitrogen dioxide, persulfate salts, permanganate salts, dichromate-sulfuric acid, hypochlorous acid, hypohalite salts, periodic acid and salts thereof, TEMPO ((2,2,6,6-tetramethylpiperidin-1-yl)oxyl), and transition metal catalysts. [0065] In various embodiments, the agent for oxidation step (i) is selected from the group consisting of periodic acid and salts thereof. Periodic acid is the highest oxoacid of iodine, with the iodine having an oxidation state of +7. Periodic acid can exist in two forms: orthoperiodic acid (HsIOe) and metaperiodic acid (HIC ), with the same being true of its salts. Salts of periodic acid are known and are not limited in respect of the counterion. Thus, in various embodiments, the agent is a salt of periodic acid. In various embodiments, the salts of periodic acid may be alkali metal salts of periodic acid; for example, sodium periodate or potassium periodate.

[0066] Periodate oxidation of 1 ,2-diols can lead to carbon-carbon bond cleavage. Such 1 ,2- diols are found in saccharides, such as those constituting the repeat units of polysaccharide biopolymers. Thus, periodate oxidation of saccharides can be used to open the saccharide ring leaving two aldehyde groups. Without wishing to be bound by theory, the periodate oxidation of 1 ,2-diols is understood to proceed by the following mechanism: wherein M is H or an alkali metal ion. Thus, in various embodiments, the ring-opening oxidation by periodate may be preferred over radical oxidation such as by TEMPO ((2, 2,6,6- tetramethylpiperidin-1-yl)oxyl).

[0067] The skilled person will be able to select suitable reaction conditions for the oxidation of the biopolymer particles according to the present disclosure. In various embodiments, the oxidation may be performed at room temperature (about 20 °C). In various embodiments, the oxidation may be performed in aqueous solution. The use of aqueous conditions may be preferable to reduce environmental impact, to aid in downstream recycling, and to avoid the need to remove traces of solvents that may be undesirable or prohibited for use in downstream applications. In various embodiments, the concentration of the oxidising agent may be present in the aqueous solution at a concentration of from about 1 to about 100 mM, from about 10 to about 90 mM, from about 20 to about 80 mM, from about 30 to about 70 mM, or from about 40 to about 60 mM.

[0068] The extent to which the oxidation reaction is allowed to proceed will determine the degree of oxidation of the biopolymer particles. As used herein, the degree of oxidation is defined as the mole percentage of generated carbonyl groups relative to the total number of structural repeats in the biopolymer. Thus, for polysaccharides such as cellulose, the degree of oxidation is defined as the mole percentage of carbonyl groups relative to the total amount of anhydrous glucose units in the biopolymer. The skilled person will be able to vary the degree of oxidation as a matter of routine optimisation of the reaction conditions.

[0069] Methods for determining the degree of oxidation are specifically within the common general knowledge of a person skilled in the art. For example, the degree of oxidation can be determined by titration. In a titration method, the biopolymer is first reacted with hydroxylamine hydrochloride. Aldehyde groups present in the biopolymer will react stoichiometrically (1 :1) with the hydroxylamine hydrochloride to form oxime groups and release hydrochloric acid. The number of moles of hydrochloric acid released is determined by titration with sodium hydroxide, with the number of moles of sodium hydroxide used at the equivalence point corresponding directly to the number of moles of aldehyde groups present in the biopolymer. A person skilled in the art will thus be able to select appropriate reaction parameters to achieve the desired degree of oxidation and monitor reaction progress accordingly. For example, reaction progress can be monitored by performing a time course wherein samples of the reaction are taken at regular intervals, quenched, and the degree of oxidation for each time point determined as detailed above. From such a reaction trajectory, the skilled person can readily determine the reaction time required to achieve a desired degree of oxidation.

[0070] In various embodiments, the reaction may be performed under mild agitation, for example by use of an overhead stirrer.

[0071] In various embodiments, the oxidised biopolymer particles are washed to remove the oxidising agent. The oxidised biopolymer articles may, for example, be washed with water. The oxidised biopolymer particles may be separated from the solvent between washing steps by sieving. Multiple washing steps may be performed, and the degree of removal of the oxidising agent may be monitored by methods known to a person skilled in the art, for example UV- visible spectrophotometry. The presence of periodate can be quantitatively determined by the measurement of the absorbance at 290 nm. In various embodiments, the oxidised biopolymer particles are washed with water until the washed particles are substantially free of oxidising agent.

[0072] The functional groups, e.g. aldehyde groups, produced by the oxidation step of the method of the first aspect provide useful reactive moieties for further functionalisation of the biopolymer particles. In the present disclosure, it has been found that lower degrees of oxidation are preferable in order to facilitate further functionalisation while retaining similar physicochemical properties as the unmodified particles. For example, too high a degree of oxidation may lead to loss of mechanical stability of the particles and disaggregation, which may impair the separability and re-use of the particles in downstream applications. Thus, in the present disclosure, the degree of oxidation of the oxidised biopolymer particles is less than or equal to about 25%. In various embodiments, the degree of oxidation of the oxidised biopolymer particles is less than or equal to about 20%, or less than or equal to about 15%.

[0073] In various embodiments, the degree of oxidation of the oxidised biopolymer particles is at least about 1% or at least about 5%. In various embodiments, the degree of oxidation of the biopolymer particles is from about 1 % to about 25%, from about 1% to about 20%, or from about 1 % to about 15%. In various embodiments, the degree of oxidation of the biopolymer particles is from about 5% to about 25%, from about 5% to about 20%, or from about 5% to about 15%.

[0074] In various embodiments, the agent for oxidation step (i) is selected from the group consisting of periodic acid and salts thereof and the degree of oxidation of the oxidised biopolymer particles is less than or equal to about 25%, less than or equal to about 20%, or less than or equal to about 15%.

[0075] In various embodiments, the agent for oxidation step (i) is selected from the group consisting of periodic acid and salts thereof and the degree of oxidation of the oxidised biopolymer particles is at least about 1% or at least about 5%.

[0076] In various embodiments, the agent for oxidation step (i) is selected from the group consisting of periodic acid and salts thereof and the degree of oxidation of the biopolymer particles is from about 1% to about 25%, from about 1% to about 20%, or from about 1 % to about 15%. In various embodiments, the agent for oxidation step (i) is selected from the group consisting of periodic acid and salts thereof and the degree of oxidation of the biopolymer particles is from about 5% to about 25%, from about 5% to about 20%, or from about 5% to about 15%.

Hydrophobic functionalisation

[0077] In the method of the first aspect, the oxidised biopolymer particles are reacted with a compound comprising one or more hydrophobic moieties (step (ii)). [0078] As used herein, “hydrophobic” takes its usual meaning in the art, namely something that is seemingly repelled from a mass of water. The terms “hydrophobic moiety” and “hydrophobic group” may be used interchangeably in the present disclosure. Hydrophobic moieties have a tendency to associate in an aqueous environment due to the exclusion of nonpolar molecules. Non-limiting examples of hydrophobic moieties include aryl, alkyl, cycloalkyl, aralkyl or alkenyl groups.

[0079] The compound comprising one or more hydrophobic moieties may, in some embodiments be a hydrocarbyl amine. In such embodiments, it will be understood by a person of ordinary skill in the art that the hydrophobic moiety is the hydrocarbyl group.

[0022] As used herein, the term “hydrocarbyl” refers to a group comprising at least C and H. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain heteroatoms. Suitable heteroatoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen, oxygen, phosphorus and silicon. Non-limiting examples of such hydrocarbyls are alkyl groups, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, and isomeric forms thereof; cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cycloocytyl, 2-methylcyclopentyl, 2,3-dimethyl- cyclobutyl, 4-methylcyclobutyl, 3-cyclopentylpropyl, and the like; cycloalkenyl groups, such as cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like, and isomeric forms thereof; cycloalkadienyl groups, such as cyclopentadientyl, cyclohexadienyl, cycloheptadienyl, and the like; aryl groups, such as phenyl, tolyl, xylyl, naphthyl, biphenylyl, and the like; aralkyl groups, such as benzyl, phenethyl, phenpropyl, naphthmethyl, and the like. Preferably, the hydrocarbyl group is an aryl, heteroaryl, alkyl, cycloalkyl, aralkyl or alkenyl group.

[0080] In various embodiments of the present disclosure, the amine group of the hydrocarbyl amine is able to react with functional groups on the oxidised biopolymer particle surface, such as aldehyde groups. The reaction of amines with aldehydes is known, typically resulting in the formation of an imine. Thus, the hydrocarbyl amine when reacted with the oxidised biopolymer particle will generally become covalently attached to the particle.

[0081] In various embodiments, the hydrocarbyl amine may be a primary hydrocarbyl amine. In further embodiments, the hydrocarbyl amine is a hydrocarbyl diamine, i.e. a hydrocarbyl group substituted with two amine groups. In such embodiments, for each hydrocarbyl diamine molecule either or both of the amino groups may react with functional groups on the oxidised biopolymer particle. In further embodiments, each amine group of the diamine is a primary amine.

[0082] In various embodiments, the hydrocarbyl group of the hydrocarbyl amine is selected from the group consisting of C6-C14 hydrocarbyl, preferably from the group consisting of Ce-Cw hydrocarbyl. Thus, in various embodiments, the hydrocarbyl amine is selected from the group consisting of Ce-Ci4 hydrocarbyl amines, preferably from the group consisting of Ce-Cw hydrocarbyl amines. In further embodiments, the hydrocarbyl amine is a hydrocarbyl diamine selected from Ce-Ci4 hydrocarbyl diamines, preferably Ce-Cw hydrocarbyl diamines.

[0083] In various embodiments, the hydrocarbyl group of the hydrocarbyl amine is an alkyl group. Thus, in various embodiments the hydrocarbyl amine is selected from the group consisting of Ce-Ci4 alkyl amines, preferably from the group consisting of Ce-Cw alkyl amines. In further embodiments, the hydrocarbyl amine is an alkyl diamine selected from the group consisting of Ce-Ci4 alkyl diamines, preferably from the group consisting of Ce-Cw alkyl diamines. In any of the above embodiments, the alkyl group may preferably be a linear alkyl group.

[0084] In some embodiments, the hydrocarbyl amine is selected from the group consisting of 1 ,6-diaminohexane; 1 ,8-diaminooctane; 1 ,10-diaminodecane; 1 ,12-diaminododecane; and 1 ,14-diaminotetradecane. In further embodiments, the hydrocarbyl amine may be selected from the group consisting of 1 ,6-diaminohexane; 1 ,8-diaminooctane; and 1 , 10-diaminodecane.

[0085] The skilled person will be able to select suitable reaction conditions for step (ii) of the method of the first aspect. In some embodiments, step (ii) is performed in aqueous solution. In some embodiments, the solvent for step (ii) is methanol. Thus, in some embodiments, the oxidised biopolymer particles may be subjected to solvent exchange, for example from water to methanol, before they are reacted with the compound comprising at least one hydrophobic moiety.

[0086] In various embodiments, step (ii) of the method of the first aspect is performed at room temperature (about 20 °C). The reaction time is not limited, and a person skilled in the art will be able to determine a suitable duration for the reaction, for example to ensure that the reaction reaches completion. [0087] In various embodiments, a molar excess of the compound comprising at least one hydrophobic moiety may be used in the reaction of step (ii). Thus, in some embodiments, the oxidised biopolymer particles may be reacted with a molar excess of about 1.2, about 1.3, about 1.4, or about 1.5 of the compound comprising one or more hydrophobic moieties. The molar excess is typically calculated on the basis of the amount of oxidised functional groups, e.g. aldehyde groups, present in the oxidised biopolymer articles.

[0088] Thus, in some embodiments, step (ii) of the method of the first aspect comprises reacting the oxidised biopolymer particles with at least about 1.2, at least about 1.3, at least about 1.4, or at least about 1.5 molar equivalents (relative to the amount of aldehyde groups) of a hydrocarbyl amine. In further embodiments, step (ii) of the method of the first aspect comprises reacting the oxidised biopolymer particles with at least about 1 .2, at least about 1 .3, at least about 1.4, or at least about 1.5 molar equivalents (relative to the amount of aldehyde groups) of a hydrocarbyl diamine, preferably wherein the hydrocarbyl diamine is selected from the group consisting of C6-C14 hydrocarbyl, more preferably wherein the hydrocarbyl diamine is selected from the group consisting of C6-C14 alkyl diamines, even more preferably wherein the hydrocarbyl diamine is selected from the group consisting of 1 ,6-diaminohexane, 1 ,8- diaminooctane, 1 ,10-diaminodecane, 1 ,12-diaminododecane, and 1 , 14-diaminotetradecane.

[0089] In further embodiments, step (ii) of the method of the first aspect comprises reacting the oxidised biopolymer particles with at least about 1 .2, at least about 1.3, at least about 1.4, or at least about 1.5 molar equivalents (relative to the amount of aldehyde groups) of a hydrocarbyl diamine, preferably wherein the hydrocarbyl diamine is selected from the group consisting of Ce-Cw hydrocarbyl diamines, more preferably wherein the hydrocarbyl diamine is selected from the group consisting of Ce-Cw alkyl diamines, still more preferably wherein the hydrocarbyl diamine is selected from the group consisting of 1 ,6-diaminohexane, 1 ,8- diaminooctane, and 1 ,10-diaminodecane.

[0090] The skilled person will understand that by using a molar excess, an (approximately) quantitative conversion of the functional groups (e.g. aldehyde groups) present on the oxidised biopolymer particles may be ensured. Thus, a specific percentage of functionalisation may be readily achieved by the quantitative conversion of oxidised biopolymer particles with a specific degree of oxidation. In this way, the degree of hydrophobic functionalisation of the biopolymer particles may be readily controlled.

[0091] Thus, in various embodiments, the hydrocarbyl amine is selected from the group consisting of Ce-Ci4 hydrocarbyl amines, preferably from the group consisting of Ce-Cw hydrocarbyl amines, and the degree of oxidation of the biopolymer particles is from about 1% to about 25%, from about 1% to about 20%, or from about 1 % to about 15%. In further embodiments, the hydrocarbyl amine is selected from the group consisting of C6-C14 alkyl amines, preferably from the group consisting of Ce-Cw alkyl amines, and the degree of oxidation of the biopolymer particles is from about 1% to about 25%, from about 1 % to about 20%, or from about 1 % to about 15%.

[0092] In various embodiments, the hydrocarbyl amine is a hydrocarbyl diamine selected from the group consisting of Ce-Ci4 hydrocarbyl diamines, preferably from the group consisting of Ce-Cw hydrocarbyl diamines, and the degree of oxidation of the biopolymer particles is from about 1% to about 25%, from about 1 % to about 20%, or from about 1% to about 15%. In further embodiments, the hydrocarbyl diamine is selected from the group consisting of Ce-Ci4 alkyl diamines, preferably from the group consisting of Ce-Cw alkyl diamines, and the degree of oxidation of the biopolymer particles is from about 1 % to about 25%, from about 1 % to about 20%, or from about 1 % to about 15%.

[0093] In various embodiments, the hydrocarbyl amine is selected from the group consisting of C6-C14 hydrocarbyl amines, preferably from the group consisting of Ce-Cw hydrocarbyl amines, and the degree of oxidation of the biopolymer particles is from about 5% to about 25%, from about 5% to about 20%, or from about 5% to about 15%. In further embodiments, the hydrocarbyl amine is selected from the group consisting of Ce-Ci4 alkyl amines, preferably from the group consisting of Ce-Cw alkyl amines, and the degree of oxidation of the biopolymer particles is from about 5% to about 25%, from about 5% to about 20%, or from about 5% to about 15%.

[0094] In various embodiments, the hydrocarbyl amine is a hydrocarbyl diamine selected from the group consisting of Ce-Ci4 hydrocarbyl diamines, preferably from the group consisting of Ce-Cw hydrocarbyl diamines, and the degree of oxidation of the biopolymer particles is from about 5% to about 25%, from about 5% to about 20%, or from about 5% to about 15%. In further embodiments, the hydrocarbyl diamine is selected from the group consisting of Ce-Ci4 alkyl diamines, preferably from the group consisting of Ce-Cw alkyl diamines, and the degree of oxidation of the biopolymer particles is from about 5% to about 25%, from about 5% to about 20%, or from about 5% to about 15%.

[0095] In any of the preceding embodiments, the agent for the oxidation step (i) may be selected from the group consisting of periodic acid and salts thereof. For example, the hydrocarbyl diamine may be selected from the group consisting of Ce-Ci4 hydrocarbyl diamines, preferably from the group consisting of Ce-C hydrocarbyl diamines, the degree of oxidation of the biopolymer particles may be from about 5% to about 25%, from about 5% to about 20%, or from about 5% to about 15%, and the agent for oxidation step (i) may be selected from the group consisting of periodic acid and salts thereof.

[0096] In further embodiments, the hydrocarbyl diamine may be selected from the group consisting of 1 ,6-diaminohexane; 1 ,8-diaminooctane; 1 ,10-diaminodecane; 1 ,12- diaminododecane; and 1 ,14-diaminotetradecane, the degree of oxidation of the biopolymer particles is from about 5% to about 25%, from about 5% to about 20%, or from about 5% to about 15%, and the agent for oxidation step (i) may be selected from the group consisting of periodic acid and salts thereof.

Reduction

[0097] In various embodiments, the method of the first aspect further comprises the step of reducing the biopolymers of step (ii) with a reducing agent. The reducing agent is not limited, and the skilled person will be able to select reducing agents appropriate to the chemistry of the group(s) being reduced.

[0098] Non-limiting examples of commonly used reducing agents include sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride. Where the compound comprising one or more hydrophobic moieties is a hydrocarbyl amine, the product of step (ii) will typically be an imine. Subsequent reaction of the imine functionalised biopolymer particles, for example with sodium borohydride, leads to reduction of the imine groups to amine groups, such that the hydrophobic moiety (e.g. hydrocarbyl group) will be attached covalently to the biopolymer particles via the amine functionality. Such a linkage may be less labile and more resistant to hydrolysis.

[0099] A person skilled in the art will be able to select appropriate reaction conditions for the reduction step. The present disclosure is not limited in this respect.

[0100] In various embodiments, the reduction step is performed at room temperature (about 20 °C). In some embodiments, the solvent for the reduction step is methanol. In some embodiments, the reduction step is performed in aqueous solution. Thus, the reduction step may be performed in the same or different solvent as step (ii) of the method of the first aspect. Where the solvent for the reduction is different to the solvent of step (ii), the biopolymer particles may be subjected to solvent exchange, for example by sieving. [0101] In various embodiments, a molar excess (relative to the number of functional groups on the biopolymer particles to be reduced) of reducing agent may be used. In various embodiments, at least about 1.2, at least about 1.5, at least about 1.8, or at least about 2.0 molar equivalents (relative to the amount of carbonyl groups) of reducing agent are used in the reduction step. When the reduction step is performed in water, a higher excess of reducing agent may be preferred when using a reducing agent such as sodium borohydride that is more susceptible to hydrolysis.

[0102] After reduction, the reduced biopolymer particles are typically washed to remove all traces of reducing agent. In various embodiments, the reduced biopolymer particles are washed with water. Where methanol is used as a solvent, the washing also serves to remove all traces of methanol from the reduced biopolymer particles.

[0103] In various embodiments of the method of the first aspect, the agent for the oxidation step (i) is selected from the group consisting of periodic acid and salts thereof, the degree of oxidation of the oxidised biopolymer particles is from about 1% to about 25%, from about 1% to about 20%, or from about 1% to about 15%, and said method further comprises the step of reducing the biopolymers of step (ii) with a reducing agent.

[0104] In various embodiments of the method of the first aspect, the agent for the oxidation step (i) is selected from the group consisting of periodic acid and salts thereof, the degree of oxidation of the oxidised biopolymer particles is from about 5% to about 25%, from about 5% to about 20%, or from about 5% to about 15%, and said method further comprises the step of reducing the biopolymers of step (ii) with a reducing agent.

[0105] In any of the preceding embodiments, the oxidised biopolymer particles are reacted in step (ii) with a hydrocarbyl diamine, preferably wherein the hydrocarbyl diamine is selected from the group consisting of Ce-Cu hydrocarbyl diamines, more preferably from the group consisting of C6-C14 hydrocarbyl diamines, even more preferably from the group consisting of C6-C14 alkyl diamines, and still more preferably from the group consisting of 1 ,6- diaminohexane; 1 ,8-diaminooctane; 1 ,10-diaminodecane; 1 ,12-diaminododecane; and 1 ,14- diaminotetradecane.

[0106] For example, in some embodiments of the method of the first aspect, the agent for the oxidation step (i) is selected from the group consisting of periodic acid and salts thereof, the degree of oxidation of the oxidised biopolymer particles is from about 5% to about 25%, from about 5% to about 20%, or from about 5% to about 15%; the oxidised biopolymer particles are reacted in step (ii) with a hydrocarbyl diamine, preferably wherein the hydrocarbyl diamine is selected from the group consisting of C6-C14 hydrocarbyl diamines, more preferably from the group consisting of C6-C14 hydrocarbyl diamines, even more preferably from the group consisting of C6-C14 alkyl diamines, and still more preferably from the group consisting of 1 ,6- diaminohexane; 1 ,8-diaminooctane; 1 ,10-diaminodecane; 1 ,12-diaminododecane; and 1 ,14- diaminotetradecane, wherein said method further comprises the step of reducing the biopolymers of step (ii) with a reducing agent.

[0107] In any of the preceding embodiments, the reducing agent may be selected from the group consisting of sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride.

Enzyme immobilisation

[0108] The functionalised biopolymer particles of the present disclosure have been found to be particularly suitable as substrates for the immobilisation of enzymes. Thus, in various embodiments the method of the first aspect further comprises the step of contacting the reduced biopolymer particles with an enzyme to form enzyme functionalised biopolymer particles.

[0109] Immobilised enzymes are routinely used for the manufacture of many industrial products, for example in the manufacture of pharmaceuticals, chemicals and foodstuffs. For example, BASF have used the lipase from Candida Antarctica (CalB) to produce chiral compounds such as dimethenamide-P, a herbicide that was previously produced by chemical synthesis.

[0110] Enzymes can be attractive catalysts in industry as they may be used at lower reaction temperatures and can stereospecifically produce chiral compounds. Moreover, enzymes may have a preferable safety and/or environmental profile over chemical reagents/catalysts. Accordingly, biocatalysis is generally considered a ‘green’ process in comparison to chemical synthesis.

[0111] As for all industrial catalysts, immobilisation, being a form of heterogeneous catalysis, brings further benefits in terms of simplified downstream processing or continuous process operations. This is because immobilised enzymes can be recovered and reused, often while retaining activity for long periods of time. Enzymes can be expensive to produce at scale and so their recovery and reuse is generally economically preferable. Further, immobilised enzymes are typically amenable to a variety of process configurations, for example in batch stirred reactors as well as in packed bed column reactors.

[0112] The functionalised biopolymer particles of the present disclosure are particularly useful supports for the immobilisation of enzymes because the biopolymer particles can be produced from renewable and biodegradable feedstock rather than petrochemicals. Further, the biopolymer particles can be produced according to the methods disclosed herein without the use of hazardous or environmentally harmful substances such as organic solvents and which facilitate the re-use and recycling of the process chemicals such as the solvent and antisolvent.

[0113] Enzymes that may be contacted with the reduced biopolymer particles of the present disclosure are not limited. Non-limiting examples of important categories of industrial enzymes are carbohydrases, proteases and lipases.

[0114] In various embodiments of the present disclosure, the reduced biopolymer particles are contacted with an enzyme which is a lipase. Lipases are enzymes that catalyse the hydrolysis of lipids. Many lipases are known and/or commercially available and the selection and/or production of lipases by conventional means is specifically within the common general knowledge of a person skilled in the art.

[0115] In various embodiments, the lipase is purified from a microorganism that endogenously produces said lipase. In various embodiments, the lipase is recombinantly produced in a heterologous microorganism. In various embodiments of the present disclosure, the lipase is selected from the group consisting of Candida antartica lipase B, Thermomyces lanuginosus lipase, and Pseudomonas cepacia lipase, all of which are commercially available, for example from Sigma-Aldrich.

[0116] As discussed above, it has been found that the reduced biopolymer particles of the present disclosure may be used for the immobilisation of enzymes. Thus, in various embodiments, upon contacting the reduced biopolymer particles with an enzyme, the enzyme is immobilised on said biopolymer particles, producing what are referred to herein as enzyme functionalised biopolymer particles.

[0117] Without wishing to be bound by theory, it is believed that the immobilisation is mediated by non-covalent interactions. The present disclosure is not limited in terms of such interactions, and the skilled person will understand that non-covalent interactions may include van der Waals interactions, hydrogen bonding, ionic interactions, halogen bonding, pi-effects such as pi-pi, cation-pi or anion-pi interactions, and combinations thereof.

[0118] The enzyme functionalised biopolymer particles of the present disclosure have been found to exhibit good levels of enzyme immobilisation, and in particular higher levels of adsorption compared with biopolymer particles that have not been functionalised by the methods disclosed herein. Moreover, the enzyme functionalised biopolymer particles of the present disclosure exhibit good retention of enzyme activity after at least two rounds of recycling. For example, in the tests reported in the Examples and Figure 5, CalB immobilised on 1 ,6-diaminohexane functionalised biopolymer particles exhibited generally stable levels of enzyme activity after an initial recycling step and up to the tenth recycling step (Figure 5a).

[0119] The enzyme functionalised biopolymer particles of the present disclosure also exhibit good storage stability even in the absence of stabilisers and/or preservatives. As used herein, storage stability refers to the retention of enzyme activity when stored in a solvent or solution, such as water. For example, the enzyme functionalised biopolymer particles of the present disclosure may exhibit less than 50%, less than 40%, or less than 30% loss of enzyme activity after storage in water for at least 1 month, at least 2 months, or at least 3 months. In various embodiments, the enzyme functionalised biopolymer particles are stored in water at 4 °C.

[0120] The enzyme immobilised biopolymer particles are particularly useful for biocatalysis, i.e. they may be used for catalysing a reaction. Thus, in one aspect, the present disclosure provides a method for performing an enzyme-catalysed reaction wherein the enzyme catalysing the reaction is immobilised on functionalised biopolymer particles prepared by the methods disclosed herein. The nature of the reaction is not limited, and the skilled person will be able to select enzymes capable of performing the desired chemical transformation. The reaction may be performed in aqueous solution or may be performed in one or more organic solvents. The selection of suitable solvent systems and other reaction conditions for performing a biocatalytic reaction is also in the common general knowledge of a person skilled in the art.

[0121] The present disclosure provides a generally applicable methodology for the functionalisation of biopolymer particles. Accordingly, the present disclosure is not limited in terms of the means by which the biopolymer particles are prepared.

[0122] Two non-limiting examples of methods by which biopolymer particles may be prepared are (i) extrusion, and (ii) membrane emulsification followed by phase inversion. To prepare biopolymer particles by extrusion, a dispersed phase is extruded into an anti-solvent to form particles of the biopolymer. The dispersed phase comprises the biopolymer in a solvent as discussed further below, and the extrusion of such a dispersed phase is known in the art. It is a process wherein the dispersed phase is forced, pressed, or pushed out, for example through an aperture or opening. The opening may be in a syringe as shown in Figure 1 or any other suitable extrusion device as known in the art.

[0123] A schematic representation of an exemplary embodiment of the extrusion process is shown in Figure 1. In the exemplary embodiment of Figure 1 , the dispersed phase (1) comprising the biopolymer in a solvent is extruded through a needle (2) of a syringe (3). Extrusion is specifically into the anti-solvent (4) to form biopolymer particles (5). In the exemplary embodiment of Figure 1 , the extruded dispersed phase is dropped from a height, d, above the surface of the anti-solvent.

[0124] The latter exemplary method of preparing biopolymer particles comprises a membrane emulsification step and a phase inversion step.

[0125] Membrane emulsification is known in the art; it is a technique in which a dispersed phase is forced through the pores of a microporous membrane directly into a continuous phase, where emulsified droplets are formed and detached at the end of the pores with a drop- by-drop mechanism. A schematic representation of a membrane emulsification process is shown in Figure 2, where the arrow indicates the direction of flow.

[0126] The dispersed phase generally includes a first liquid containing the biopolymer dissolved in a solvent, and the continuous phase includes a second liquid which is immiscible with the first liquid. The interaction of the two liquids when the dispersed phase is pushed or otherwise transported through the membrane is called a dispersion process, and their inhomogeneous mixture is termed an emulsion, i.e. droplets of the dispersed phase surrounded by the continuous phase.

[0127] In the context of producing biopolymers, the droplets of dispersed phase in continuous phase have been successfully isolated by phase inversion. In the context of cellulose, this is described in ACS Sustainable Chem. Eng. 2017, 5, 7, 5931-5939, which is incorporated herein by reference. Phase inversion is a chemical phenomenon exploited in the fabrication of artificial membranes, and is performed by removing solvent from a liquid-polymer solution. There are various methods of phase inversion including immersing the polymer solution into a third liquid called the anti-solvent. The use of anti-solvent based phase inversion has proven to be particularly effective in precipitating droplets of biopolymer into particles from an emulsion of dispersed/continuous phase.

[0128] Common to both exemplary processes for preparing biopolymer particles is the use of a solvent into which the biopolymer is dissolved to form the dispersed phase, and the use of an anti-solvent to form the biopolymer particles.

[0129] Solvents for use in the preparation of biopolymer particles, particularly by membrane emulsification or extrusion, are known and ionic liquids are commonly favoured as they are able to solubilise recalcitrant biopolymers. Ionic liquids are salts that are in liquid form at a temperature between ambient temperature and 100°C, for example imidazolium based ionic liquids such as 1-ethyl-3-methylimidazolium acetate (EmimOAc), 1-butyl-3-methylimidazolium chloride (BmimOAc) or the like. Moreover, ionic liquids are essentially non-volatile (avoiding fugitive emissions) and are considered to have environmental benefits over other solvents. Ionic liquids can, for example, be readily recycled by distillation to remove the anti-solvent.

[0130] Ionic liquids are typically not used in pure form, however. An amount of a co-solvent is often added to the ionic liquid when dissolving biopolymers such as cellulose. The use of a cosolvent may assist in dissolution of the biopolymer, and may reduce the amount of costly ionic liquid required. In methods for forming biopolymer particles, the inclusion of a co-solvent may also improve the efficiency and yield of the process by modifying the viscosity of the dispersed phase, which may in turn reduce the amount of deformation exhibited by the particles.

[0131] Typical co-solvents employed in combination with ionic liquids are dipolar aprotic solvents, such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and the like. However, such solvents are not generally considered to be environmentally friendly, and the use thereof may therefore have a negative impact on the overall environmental benefits of ‘green’ processes that use ionic liquids. Notably, DMSO is listed in Annex II of Regulation (EC) No. 1223/2009 on Cosmetic Products (available at https://echa.europa.eu/cosmetics- prohibited-substances), and DMF is associated with toxic effects. Such co-solvents therefore cannot be used in processes for the preparation of biopolymer particles for use in cosmetic and personal care as well as other applications. The use of dipolar aprotic solvents may also complicate the recycling of the ionic liquid and increase costs. For example, some degree of distillation of DMSO is to be expected during recycling and the presence of aprotic solvent has been reported to reduce the thermal stability of 1-ethyl-3-methylimidazolium acetate (EmimOAc) [see Williams et al., Thermochimica Acta (2018), 669 126-139, for example]. [0132] T urning now to the anti-solvents that may be used when preparing biopolymer particles, organic solvents such as ethanol are typical. Again, however, these substances may reduce the overall environmental benefits of the process, may be associated with safety concerns, and may complicate and increase the cost of recycling of ionic liquids.

[0133] Thus, in various embodiments, an aqueous solvent and an aqueous anti-solvent may be used. The use of an aqueous solvent and anti-solvent may obviate the use of reagents associated with environmental and safety concerns and may also simplify and reduce the cost of solvent recycling. In particular, use of such solvents and anti-solvents may increase the stability of ionic liquids against temperature-based degradation [Williams et al., Thermochimica Acta (2018), 669: 126-139], which may allow an increased number of recycling cycles to be performed, for example. Finally, the inclusion of water in the dispersed phase may increase the likelihood of bead sphericity.

[0134] Accordingly, in various embodiments, the dispersed phase from which the biopolymer particles may be prepared comprises a solvent in which the biopolymer is dispersed or dissolved, which solvent comprises water. By the term “solvent” is therefore meant any substance (e.g. liquid) which disperses or dissolves the biopolymer. The term “solvent” also includes solvent mixtures.

[0135] The solvent of the dispersed phase may comprise water and may comprise an ionic liquid, an organic solvent, an inorganic nonaqueous solvent, or a combination thereof. In various embodiments of the present disclosure, the solvent for the dispersed phase comprises water and at least one of an ionic liquid, an organic solvent, an inorganic nonaqueous solvent, or a combination thereof. In various embodiments of the present disclosure, the solvent for the dispersed phase comprises water and one or more ionic liquid(s).

[0136] Non-limiting examples of solvents for the dispersed phase other than water include methanol, ethanol, ammonia, acetone, acetic acid, n-propanol, n-butanol, isopropyl alcohol, ethyl acetate, dimethyl sulfoxide, sulfuryl chloride, phosphoryl chloride, carbon disulfide, morpholine, N-methylmorpholine, NaOH without and with association of urea and thiourea, bromine pentafluoride, hydrogen fluoride, sulfuryl chloride fluoride, acetonitrile, dimethylformamide, hydrocarbon oils and blends thereof, toluene, chloroform, carbon tetrachloride, benzene, hexane, pentane, cyclopentane, cyclohexane, 1 ,4-dioxane, dichloromethane, nitromethane, propylene carbonate, formic acid, tetrahydrofuran, diethyl ether, phosphoric acid, 1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride, 1-methoxymethyl-3-methylimidazolium bromide, N-ethylpyridinium chloride, N- methylmorpholine-N-oxide, 1-methylimidazole, N,N-dimethylformamide, N,N'- dimethylimidazolidin-2-one, N,N-dimethylacetamide, sulfolane, y-valerolactone, y- butyrolactone, N,N,N',N'-tetramethylurea, N-methylpyrrolidinone, and methylene chloride. The skilled person will readily recognise which of the exemplary solvents are ionic liquids, organic solvents, and/or inorganic non-aqueous solvents.

[0137] As will be understood by the skilled person in the art, the dispersed phase will depend on the biopolymer being used. The identification of suitable solvents for the dispersed phase of the present disclosure is specifically within the common general knowledge of the skilled person.

[0138] In various embodiments, the solvent for the dispersed phase comprises water and an ionic liquid. The ionic liquid may be selected from 1-ethyl-3-methylimidazolium acetate (EmimOAc), 1-butyl-3-methylimidazolium chloride (BmimOAc), and a combination thereof. In some embodiments, the solvent for the dispersed phase comprises water and one or more organic solvents. In other embodiments, the solvent for the dispersed phase is substantially free of organic solvents. The term “substantially free” is defined above. The skilled person will understand that when the solvent of the dispersed phase consists of water and an ionic liquid, the total wt% of water and ionic liquid in the dispersed phase solvent will total 100 wt%. If water is present, for example, in an amount of at least 0.5 wt%, an ionic liquid may be present in an amount of at least 99.5 wt%, with the proviso that the total of water and ionic liquid is 100 wt%. In other words, the ionic liquid may be present as the remainder of the solvent.

[0139] Preferably, the solvent used for the dispersed phase is environmentally friendly. By the term “environmentally friendly” is meant not harmful to the environment such that the solvent can be disposed of without the need for specialist equipment or process(es), i.e. non-toxic. It is known in the art that polysaccharides have limited dissolution in most of the common solvents. It is also known in the art that those solvents which do dissolve polysaccharides are often toxic and/or highly selective. When the biopolymer is a polysaccharide such as cellulose, starch, chitin, glycogen, and/or chitosan, the solvent for the dispersed phase may therefore comprise an ionic liquid in addition to water. The dissolution of cellulose with the ionic liquid 1- butyl-3-methylimidazolium chloride is, for example, discussed in Richard et al., J. Am. Chem. Soc. 2002, 124, 4974-4975. Verma et al., Sustainable Chemistry and Pharmacy 13 (2019), 100162 similarly discusses the solubility of cellulose in ionic liquids and ionic liquids with cosolvents. Each of these disclosures is incorporated herein by reference. [0140] The concentration of biopolymer in the dispersed phase is not limited and may be any concentration suitable for the methods discussed herein. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 0.1 wt% to about 15 wt%. In various embodiments the biopolymer is present in the dispersed phase in an amount from about 0.5 wt% to about 15 wt%. In various embodiments the biopolymer is present in the dispersed phase in an amount from about 1 wt% to about 15 wt%. In various embodiments the biopolymer is present in the dispersed phase in an amount from about 1.5 wt% to about 15 wt%. In various embodiments the biopolymer is present in the dispersed phase in an amount from about 2 wt% to about 15 wt%. In various embodiments the biopolymer is present in the dispersed phase in an amount from about 2.5 wt% to about 15 wt%. In various embodiments the biopolymer is present in the dispersed phase in an amount from about 3 wt% to about 15 wt%. In various embodiments the biopolymer is present in the dispersed phase in an amount from about 3.5 wt% to about 15 wt%. In various embodiments the biopolymer is present in the dispersed phase in an amount from about 4 wt% to about 15 wt%.

[0141] In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 0.1 wt% to about 12 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 0.5 wt% to about 12 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 1 wt% to about 12 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 1.5 wt% to about 12 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 2 wt% to about 12 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 2.5 wt% to about 12 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 3 wt% to about 12 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 3.5 wt% to about 12 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 4 wt% to about 12 wt %.

[0142] In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 0.1 wt% to about 10 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 0.5 wt% to about 10 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 1 wt% to about 10 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 1.5 wt% to about 10 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 2 wt% to about 10 wt%. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 2.5 wt% to about 10 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 3 wt% to about 10 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 3.5 wt% to about 10 wt %. In various embodiments, the biopolymer is present in the dispersed phase in an amount from about 4 wt% to about 10 wt %.

[0143] The dispersed phase may further include optional components. These optional components include, but are not limited to, surfactants, porogens, active ingredients, pockets of air, double emulsions, pigments, and dyes. The level of any of the optional components is not significant in the present disclosure. In various embodiments, the dispersed phase includes a co-solvent.

[0144] The surfactant may be any suitable surfactant known in the art, for example, any ionic or non-ionic surfactant. Ionic surfactants may include sulfates, sulfonates, phosphates and carboxylates such as alkyl sulfates, ammonium lauryl sulfates, sodium lauryl sulfates, alkyl ether sulfates, sodium laureth sulfate and sodium myreth sulfate, dioctyl sodium sulfosuccinate, perfluorooctanesulfonate, perfluorobutanesulfonate, alkyl benzene sulfonates, alkyl aryl ether phosphates, alkyl ether phosphates, and alkyl carboxylates. Non-ionic surfactants may include polyethers, polyoxyalkylene derivatives of hexitol, partial long-chain fatty acid esters such as sorbitan oleates, ethylene oxide derivatives of long-chain alcohols, ethoxylated vegetable oil, polydimethylsilxoxanes, and ethylene oxide/propylene oxide copolymers.

[0145] The temperature of the dispersed phase is not limited. By the expression “temperature of the dispersed phase” or “the dispersed phase is at a temperature of”, or the like, is meant the temperature of the dispersed phase prior to extrusion or membrane emulsification (e.g. when it is placed in the apparatus for such extrusion or emulsification), and/or the temperature of the apparatus during extrusion or emulsification of the dispersed phase. As discussed in more detail below, the extrusion or emulsification means may be heated so that the dispersed phase remains at an elevated temperature in situ. Preferably the extrusion means is heated directly by one or more heating means. This is discussed further below.

[0146] In some embodiments, the dispersed phase is at ambient or room temperature, namely between about 20 and about 25°C. In various embodiments the dispersed phase is heated above ambient temperature. The dispersed phase may be heated using any suitable means. The dispersed phase is preferably heated in situ such that there is no temperature loss prior to extrusion or membrane emulsification, for example by heating a vessel containing the dispersed phase and/or the extrusion or emulsification means. In the extrusion process, a heated syringe and/or needle may, for example, be used. Suitable heating apparatus may comprise a heating element, for example a Peltier element, as well as a means of regulating the temperature, such as a thermocouple and controller.

[0147] Thus, in various embodiments, the temperature of the dispersed phase is from about 5°C to less than about 100°C, from about 10°C to less than about 100°C, from about 15°C to less than about 100°C, from about 20°C to less than about 100°C, from about 25°C to less than about 100°C, or from about 30°C to less than about 100°C. The maximum temperature will be set by the point at which the evaporation of water from the dispersed phase becomes prohibitive and/or decomposition of the ionic liquid begins to occur. This will readily be determined by the person skilled in the art.

[0148] In various embodiments, the temperature of the dispersed phase is from about 5°C to about 90°C, from about 10°C to about 90°C, from about 15°C to about 90°C, from about 20°C to about 90°C, from about 25°C to about 90°C, or from about 30°C to about 90°C. In various embodiments, the temperature of the dispersed phase is from about 5°C to about 80°C, from about 10°C to about 80°C, from about 15°C to about 80°C, from about 20°C to about 80°C, from about 25°C to about 80°C, from about 30°C to about 80°C, or from about 40°C to about 80°C.

Anti-solvent

[0149] The anti-solvent may comprise water, i.e. it may be aqueous. In various embodiments, the anti-solvent may comprise water and an organic solvent such as an alcohol or acetone, or any other organic solvent known in the art. Suitable alcohols include ethanol and/or methanol. Preferably, the anti-solvent is environmentally friendly. More preferably, the solvent and antisolvent are both environmentally friendly. Thus, in various embodiments, the anti-solvent is substantially free of organic solvents. In various embodiments, the anti-solvent is or consists of water.

[0150] In various embodiments, the anti-solvent further comprises an ionic liquid. In some embodiments, the anti-solvent may comprise water and an ionic liquid before phase inversion or extrusion of the dispersed phase. In other embodiments, the ionic liquid may be introduced into the anti-solvent during the phase inversion or extrusion. In some embodiments where the dispersed phase comprises an ionic liquid, the ionic liquid may be introduced into the antisolvent from the dispersed phase during the phase inversion or extrusion process.

[0151] In various embodiments, the concentration of ionic liquid in the anti-solvent is up to about 50 wt% - the term “up to” being understood to mean greater than zero. In various embodiments the concentration of ionic liquid in the anti-solvent is up to about 40 wt%. In various embodiments the concentration of ionic liquid in the anti-solvent is up to about 30 wt%. In various embodiments the concentration of ionic liquid in the anti-solvent is up to about 20 wt%. In various embodiments the concentration of ionic liquid in the anti-solvent is up to about 10 wt%.

[0152] Where the anti-solvent comprises water and an ionic liquid, the ionic liquid may be 1- ethyl-3-methylimidazolium acetate (EmimOAc), 1-butyl-3-methylimidazolium chloride (BmimOAc), or mixtures thereof. In various embodiments, the ionic liquid is 1-ethyl-3- methylimidazolium acetate (EmimOAc).

[0153] The temperature of the anti-solvent is not limited. In various embodiments, the temperature of the anti-solvent is from about 5°C to about 80°C. In various embodiments, the temperature of the anti-solvent is from about 10°C to about 70°C. In various embodiments the temperature of the anti-solvent is from about 15°C to about 60°C.

[0154] In the membrane emulsification process discussed herein, the temperature of the antisolvent may be ambient such that phase inversion is carried out at ambient temperature, namely between about 20 and about 25°C. In such embodiments, the anti-solvent has a temperature between about 20 and about 25°C. Alternatively and preferably, the anti-solvent is cooled to a temperature below ambient temperature, namely below about 20°C. For example, the anti-solvent may be cooled to a temperature T2, for the phase inversion (b), T2 being less than Tdisp. Preferably T2 is substantially equal to T1, more preferably T2 is equal to T1 , where T1 is defined above.

[0155] The advantage of controlling the temperature of the anti-solvent (T2) in such embodiments is to prevent pre-mature thawing of the frozen droplets. Without wishing to be bound by any one theory, it is believed that by cooling the anti-solvent to T2, the droplets remain in a frozen state (and hence spherical and non-aggregated) whilst the continuous phase surrounding them is stripped away by the phase inversion. The anti-solvent is able to contact the surface of the droplets, causing precipitation of the biopolymer and hardening of the precipitate surface. Additionally, as the frozen dispersed phase droplet thaws, the anti-solvent will convert the droplet of dissolved biopolymer to a bead/particle thereof, whilst leaching the solvent system into the anti-solvent.

(i) Extrusion

[0156] As discussed above, while the present disclosure is not limited by the means by which the biopolymer particles are prepared, a non-limiting example of a suitable method is wherein the dispersed phase is extruded into the anti-solvent to form particles of the biopolymer.

[0157] In various embodiments, the dispersed phase is extruded through a fluid medium by capillary extrusion. The fluid medium may, for example, be air. Examples of capillaries through which the dispersed phase may be extruded are glass capillaries, microfluidic channels, and (hypodermic) needles. The material from which such capillaries are prepared is not limited and the skilled person will be able to select suitable capillaries compatible with the dispersed phase.

[0158] The surface of the capillary may also be modified. The capillary may, for example, be treated, coated, or lined, in order to alter its wetting properties. Such modifications of the capillary material may, for example, alter the hydrophilicity/hydrophobicity of the capillary material, thereby altering the wettability of the capillary surface. Capillaries may, for example, be treated with reactive hydrophobic compounds such as silanes to form a hydrophobic surface layer, or hydrophobic compounds may be deposited onto a capillary surface by methods such as chemical vapour deposition. In another example, metal needles may be lined with PTFE (polytetrafluoroethylene). The identification of suitable surface modifications is specifically within the common general knowledge of the skilled person.

[0159] The size of the aperture or opening, e.g. the diameter of the capillary or the gauge of the needle, is not limited. It will be immediately apparently to a person skilled in the art that the size of the aperture or opening will, however, influence the size of the droplets of the dispersed phase extruded therefrom. Generally, a larger aperture or opening would be expected to produce larger droplets of the dispersed phase, and conversely a smaller aperture or opening would be expected to produce smaller droplets of the dispersed phase. The skilled person will be able to select appropriately sized openings/apertures.

[0160] The diameter of the aperture or opening through which the dispersed phase is extruded may be less than about 3 mm, less than about 2.5 mm, less than about 2 mm, less than about 1.5 mm, less than about 1 mm, less than about 0.75 mm, less than about 0.5 mm, less than about 0.4 mm, less than about 0.3 mm, or less than about 0.2 mm. In various embodiments, the diameter of the aperture or opening through which the dispersed phase is extruded may be greater than about 0.1 mm. In various embodiments, the diameter of the aperture or opening through which the dispersed phase is extruded may be greater than about 0.1 mm and less than about 3 mm, greater than about 0.1 mm and less than about 2.5 mm, greater than about 0.1 mm and less than about 2 mm, greater than about 0.1 mm and less than about 1.5 mm, greater than about 0.1 mm and less than about 1 mm, greater than about 0.1 mm and less than about 0.75 mm, greater than about 0.1 mm and less than about 0.5 mm, greater than about 0.1 mm and less than about 0.4 mm, or greater than about 0.1 mm and less than about 0.3 mm. In other embodiments, the diameter of the aperture or opening through which the dispersed phase is extruded may be from about 0.1 mm to about 1 mm, from about 1 mm to about 2 mm, or from about 2 mm to about 3 mm.

[0161] In various embodiments, the dispersed phase is extruded through a needle. The needle may be blunt-tipped, although the present disclosure is not limited in this respect. In various embodiments, the needle gauge size is 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , or 10 gauge.

[0162] The rate of extrusion is not limited and may be controlled using standard laboratory equipment, for example a syringe pump. In various embodiments, the rate of extrusion is less than about 1 mL/min, less than about 100 pL/min, less than about 10 pL/min, less than about 1 pL/min, or less than about 100 nL/min. In other embodiments, the rate of extrusion is from about 1 pL/min to about 1 mL/min, or from about 10 pL/min to about 100 pL/min.

[0163] In some embodiments, the dispersed phase is first extruded through a fluid medium into a mould and then the extruded dispersed phase is contacted with the anti-solvent. In various embodiments, the mould may impart a shape to the biopolymer particles formed upon contacting the extruded dispersed phase with the anti-solvent. The shape of the biopolymer particles is not limited, and will be determined by the shape of the mould in this instance. The mould may be formed of any suitable material that is compatible with the dispersed phase and anti-solvent, and may, for example, be a silicone polymer such as polydimethylsiloxane (PDMS). The mould may be prepared by casting the mould material, or may be prepared by 3D printing the mould material. The extruded dispersed phase may be contacted with the antisolvent by submerging the mould containing the extruded dispersed phase in the anti-solvent. The mould may be removed after the biopolymer particles have formed, or may be retained during further processing steps, such as washing and filtration/extraction of the biopolymer particles. [0164] When a mould is not used, extrusion may occur within the anti-solvent; that is to say, the dispersed phase may be exposed to the anti-solvent immediately upon extrusion (for example where the aperture or opening is submerged in the anti-solvent). Alternatively, and preferably, in various embodiments the extruded dispersed phase is dropped from a height above the surface of the anti-solvent. This can be seen in Figure 1 , wherein the extruded dispersed phase is dropped from a height, d, above the surface of the anti-solvent.

[0165] The dropping height may influence the sphericity of the particles obtained by the extrusion process. Without wishing to be bound by any one theory, it is believed that a greater dropping height may minimize tailing (i.e. improve sphericity) by allowing more time for cohesive forces to act on the falling droplet. Thus, in various embodiments, the extruded phase is dropped from a height of at least 10 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 20 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 30 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 40 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 50 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 60 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 70 cm above the surface of the anti-solvent, or at least 80 cm above the surface of the anti-solvent.

[0166] The maximum dropping height will be determined by the distance at which non- spherical particles are formed. This is known in the art and readily understood by the skilled person. It may, for instance, be determined by eye. In various embodiments, however, the extruded phase is dropped from a height of less than 80 cm above the surface of the antisolvent. In various embodiments, the extruded phase is dropped from a height of less than 70 cm above the surface of the anti-solvent. In various embodiments, the extruded phase is dropped from a height of In various embodiments, the extruded phase is dropped from a height of less than 60 cm above the surface of the anti-solvent. In various embodiments, the extruded phase is dropped from a height of less than 50 cm above the surface of the anti-solvent.

[0167] In various embodiments the extruded phase is dropped from a height of about 1 cm to about 80 cm above the surface of the anti-solvent, preferably from a height of about 5 cm to about 70 cm, more preferably from a height of about 10 cm to about 60 cm. [0168] In various embodiments the extruded phase is dropped from a height of about 10 cm to about 80 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of about 10 cm to about 70 cm above the surface of the antisolvent. In various embodiments the extruded phase is dropped from a height of about 10 cm to about 60 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of about 10 cm to about 50 cm above the surface of the antisolvent.

[0169] In various embodiments the extruded phase is dropped from a height of about 20 cm to about 80 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of about 20 cm to about 70 cm above the surface of the antisolvent. In various embodiments the extruded phase is dropped from a height of about 20 cm to about 60 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of about 20 cm to about 50 cm above the surface of the antisolvent. In various embodiments the extruded phase is dropped from a height of about 20 cm to about 40 cm above the surface of the anti-solvent.

[0170] The extrusion process may further comprise the step of separating the biopolymer particles from the anti-solvent. The means by which the biopolymer particles may be separated from the anti-solvent are not limited and will be known to a person skilled in the art. For example, in various embodiments, the biopolymer particles may be separated from the antisolvent by a filtration process. The filtration process is not limited and may involve mechanical or any other type of filtration (e.g. using equipment known in the art such as a hydrocyclone). In various embodiments, a filtration medium (e.g. a filter) may be used to filter the biopolymer particles from the anti-solvent and thereby collect the biopolymer particles.

[0171] In various embodiments, the biopolymer particles may be allowed to settle in a vessel and anti-solvent removed or decanted to leave biopolymer particles wetted in residual antisolvent. Alternatively, the biopolymer particles may be separated by a centrifugal separator or a disk stack separator.

[0172] In various embodiments, the biopolymer particles may be washed one or more times, for example with an aqueous solvent including water. Such washing steps may be performed to remove residual ionic liquid that may be present. In various embodiments, the solvent in which the biopolymer particles are immersed may be exchanged for an alternative solvent. (ii) Membrane emulsification

[0173] As discussed above, another non-limiting example of a suitable method for preparing biopolymer particles is membrane emulsification followed by phase inversion. Membrane emulsification involves passing a dispersed phase through a membrane into a continuous phase so as to form an emulsion. The membrane is not limited; it can be any porous structure suitable for a membrane emulsification process. For example, the membrane may be a plate with holes acting as pores (e.g. micron-sized holes), a perforated metal tube, or sintered porous glass.

[0174] By the term “emulsion” is meant the class of two-phase systems of matter where both phases are liquid. Emulsions are a type of colloid, and generally consist of two immiscible liquids. In various embodiments, the emulsion may be a macro-emulsion; this is an emulsion in which the particles of the dispersed phase have diameters of approximately 1 to 1000 microns. The term “sol” refers to a general class of two-phase systems of matter where the continuous phase is liquid and the dispersed phase is solid.

[0175] The membrane emulsification is also not limited and may be any membrane emulsification process known in the art. For example, the membrane emulsification process may be a cross-flow membrane emulsification, a rotational membrane emulsification, a vibrational membrane emulsification, or a combination thereof. As is understood in the art, the terms “cross-flow”, “rotational” and “vibrational” refer to the method used to generate shear on the membrane surface. A continuous phase could, for example, move relative to a stationary membrane to create shear, or the membrane could move relative to stationary phases. Alternatively, the dispersed phase could be injected into a stationary continuous phase. Known process parameters such as membrane type, average pore size and porosity, crossflow velocity, transmembrane pressure and emulsifier may also be used. In various embodiments, the membrane emulsification may involve a cross flow system, a stirred-cell tube membrane, a stirred cell-flat membrane, a rotating flat membrane, a vibrating/rotating tube membrane and/or a premixed membrane emulsification.

[0176] International Patent Application No. WO 01/45830 describes an example of a rotational membrane emulsification. International Patent Application No. WO 2012/094595 describes an example of a cross-flow membrane emulsification. Pedro S. Silva et al., “Azimuthally Oscillating Membrane Emulsification for Controlled Droplet Production", AIChE Journal 2015 Vol. 00, No. 00, describes a vibrational membrane emulsification: specifically a membrane emulsification system comprising a tubular metal membrane which is periodically azimuthally oscillated in a gently cross flowing continuous phase. WO 2019/092461 describes a cross-flow membrane emulsification. Each of these method descriptions is incorporated herein by reference.

[0177] In various embodiments, the membrane emulsification is a cross-flow membrane emulsification. Preferably an emulsification process in which the continuous phase moves relative to a stationary membrane.

[0178] As will be understood by the skilled person in the art, the dispersed phase and continuous phase will depend on the biopolymer being used. Various features of the solvent for the dispersed phase have already been discussed above, and said features individually or in any combination thereof are combinable with the embodiments disclosed herein. The continuous phase will comprise a solvent which is immiscible with the dispersed phase such that an emulsion is formed when the dispersed phase is forced through the porous membrane. The term “solvent” has the meaning as already defined hereinabove.

[0179] The two phases - namely the dispersed phase and the continuous phase - must be immiscible with one another. It therefore follows that the solvents for each of the phases must be immiscible with one another. The identification of suitable solvents for the dispersed phase and continuous phase of the second aspect is specifically within the common general knowledge of the skilled person.

[0180] The solvent of the continuous phase is not limited other than it must be immiscible with the dispersed phase. The solvent of the continuous phase may be a non-polar solvent. In various embodiments, the solvent of the continuous phase may be selected from hydrocarbon oils and blends thereof. Such hydrocarbon oils may be mineral oils, vegetable oils, or synthetic oils. The solvent of the continuous phase may further comprise water and/or one or more ionic liquids that may be present in residual amounts. Such residues of water and/or ionic liquid may arise as a result of solvent recycling processes.

[0181] Preferably the solvent used for the continuous phase is environmentally friendly. More preferably the solvent used for both the dispersed phase and continuous phase is environmentally friendly. The term “environmentally friendly” has the meaning as already defined hereinabove.

[0182] The continuous phase may further include optional components. These optional components include, but are not limited to, co-solvents, surfactants, pigments, and dyes. The level of any of the optional components is not significant in the present disclosure. In various embodiments, the continuous phase includes a co-solvent.

[0183] The co-solvent is not limited and may be any solvent known in the art. In various embodiments, the co-solvent may be selected from hydrocarbon oils and blends thereof. Such hydrocarbon oils may be mineral oils, vegetable oils, or synthetic oils. The co-solvent may further be a co-solvent mixture.

[0184] The surfactant is as defined above.

[0185] In various embodiments, the emulsion is cooled to a temperature Ti, Ti being greater than the pour point of the continuous phase (T _CO nt), and equal to or less than a transition temperature selected from the group consisting of the freezing point, glass transition temperature and pour point, of the dispersed phase (Tdisp): wherein Tdisp > T _CO nt. The absolute value of Ti is not, however, critical to the present disclosure; rather it is the relationship of Ti to the respective temperatures of the dispersed phase and continuous phase that is important.

[0186] The term “pour point” refers to the temperature below which a substance (e.g. liquid) loses its flow characteristics. It is typically defined as the minimum temperature at which the liquid (e.g. oil) has the ability to pour down from a beaker. The pour point can be measured with standard methods known in the art. ASTM D7346, Standard Test Method for No Flow Point and Pour Point of Petroleum Products and Liquid Fuels may, for example be used. For commercially available materials, the pour point is often provided by the supplier or manufacturer.

[0187] The term “freezing point” refers to the temperature at which a substance changes state from liquid to solid at standard atmospheric pressure (1 atmosphere). The freezing point can be measured with standard methods known in the art. ASTM E794, Standard Test Method for Melting and Crystallization Temperatures by Thermal Analysis may, for example, be used. For commercially available materials, the freezing point may be provided by the supplier or manufacturer.

[0188] The term “glass transition point” or “glass transition temperature” refers to the temperature at which a polymer structure transitions from a hard or glassy material to a soft, rubbery material. This temperature can be measured by differential scanning calorimetry according to the standard test method: ASTM E1356, Standard Test Method for Assignment of the Glass Transition Temperature by Differential Scanning Calorimetry. For commercially available materials, the glass transition temperature may be provided by the supplier or manufacturer.

[0189] Since deformation and aggregation are believed to take place when dispersed phase droplets are in a liquid state, the cooling of the emulsion to or below the pour point of the dispersed phase is believed to temporarily change - at least partially - the emulsion’s “colloid class” from an emulsion - i.e. liquid-in-liquid - to a sol - solid-in-liquid - and thereby result in the dispersed phase being easier to work with in downstream processes.

[0190] In addition, the dispersed phase having a transition temperature - the transition temperature being selected from the group consisting of freezing point, glass transition temperature and pour point - which is higher than the continuous phase pour point, means that the continuous phase surrounding the solidified dispersed phase is still able to function as a transport medium. A diagrammatic representation of an emulsion undergoing cooling and temporary conversion to a sol within a cooling coil heat exchanger is shown in Figure 2(b).

[0191] The method of cooling is not also limited. The emulsion may be cooled by any means known in the art for removing heat (energy) from a system. The emulsion may further be cooled at any point prior to phase inversion. In various embodiments, this means the emulsion is cooled simultaneously with or separately from the membrane emulsification process. The emulsion may, for example, be cooled as it is formed (e.g. by a cooling means located at the outlet of the membrane). Alternatively, the emulsion may be cooled in a step following membrane emulsification, e.g. in a cooling apparatus separate from the membrane emulsification apparatus. Advantageously, the cooling should take place as soon as possible after the emulsification takes place in order to reduce the possibility of liquid state dispersed phase droplets coalescing and/or aggregating.

[0192] In various embodiments, the emulsion may be cooled by a cooling medium (e.g. water, ice etc.) at least partially surrounding the vessel where the emulsion is formed. In a preferred embodiment, the vessel (e.g. pipe) where the emulsion is formed may have a cooling jacket containing a cooling medium. The cooling medium is not limited, and includes any medium having a lower temperature than the emulsion.

[0193] In various embodiments the emulsion may be cooled by a cooling apparatus connected to the membrane emulsification unit. The cooling apparatus may be a heat exchanger, such as an immersion heat exchanger. In an exemplary embodiment, a coil heat exchanger is immersed in a cooling medium (e.g. a cold water bath) but the disclosure is not limited in this respect. Any type of heat exchanger could, for instance, be used such as a tube-and-shell heat exchanger, a plate-and-frame heat exchanger, or a jacketed tube. Additionally, an immersion heat exchanger could be used with another cooling medium such as anti-freeze, dry ice or the like, in order to cool the emulsion to Ti.

[0194] The temperature of the anti-solvent during phase inversion is discussed above.

[0195] In various embodiments of the present disclosure, phase inversion is carried out under shear; the skilled person will be aware of suitable shear conditions for phase inversion. Shear may, for example, be achieved through the use of a stirred vessel (e.g. a mechanically stirred vessel) or a settling vessel (e.g. a gravity settling vessel). The term “shear” is used herein to refer to an external force acting on an object or surface parallel to the slope or plane in which it lies, the stress tending to produce strain.

[0196] In various embodiments, phase inversion comprises a filtration process. The filtration process is not limited and may involve mechanical or any other type of filtration (e.g. using equipment known in the art such as a hydrocyclone). A filtration process may also be encompassed by the phase inversion being carried out under shear as described above. In various embodiments, a filtration medium (e.g. filter) may be used to filter the emulsion through the anti-solvent and thereby collect the biopolymer particles. In such embodiments, the emulsion may gravity settle (shear) through the anti-solvent and into the filter, whilst the continuous phase passes through the filter (the filtrate). The frozen droplets may then be collected in the filter as the filter cake.

[0197] If not collected as part of phase inversion (e.g. via filtration or otherwise), the biopolymer particles may be separated from the anti-solvent/continuous phase mixture or the anti-solvent/continuous phase mixture may be removed from the particles. The method of removal is not limited. In various embodiments, however, the method of removal depends on whether the method is being operated in batch or continuous mode.

[0198] When the method of the second aspect is being operated in batch mode, the phase inversion step may first be performed in a closed vessel and the resulting mixture then transferred into a decanter vessel and allowed to reach a settled stage. Once settled, layers may be removed sequentially from the bottom of the vessel. Typically the order of the layers can be (1) continuous phase, (2) an interfacial layer comprising wetted biopolymer particles and (3) the remaining anti-solvent. The disclosure is not, however, limited in this respect and the skilled person will appreciate that the order of the layers will depend on their respective densities.

[0199] In various embodiments, the method is continuous and to operate in continuous mode, the phase inversion step may be performed under continuous input of emulsion and antisolvent and continuous output of the multi-phase mixture to a decanter. Within the decanter, a steady-state partition of the mixture may exist and there can be a continuous and preferably simultaneous removal from each of the phases. For example, there may be continuous and preferably simultaneous removal from: (1) the continuous phase, (2) anti-solvent and (3) wetted biopolymer particles. The order of these layers will of course vary and the method is not limited to any particular order.

[0200] Alternatively, the multi-phase (e.g. three phase) mixture may be separated using techniques known in the art, such as a disc stack separator (e.g. a centrifugal separator such as the one manufactured by Andritz).

[0201] To provide continuous cooling alongside a continuous phase inversion, the cooling medium (e.g. a medium surrounding the vessel containing the emulsion or used with a heat exchanger connected to the membrane emulsification unit) may need to be recycled or recirculated with a suitable device. A device such as a recirculating chiller (ThermoFlex available from ThermoFisher Scientific) may, for example, be used to keep the cooling medium at the desired temperature.

[0202] In various embodiments, phase inversion is followed by or involves removal of the biopolymer particles as described above. Phase inversion may be followed by decanting and then biopolymer particle removal from the mixture and/or phase inversion may involve mechanical filtration of the wetted particles from the anti-solvent/continuous phase/particle mixture.

[0203] Alternatively, the biopolymer particles may be removed from the continuous phase before phase inversion. In such embodiments, wetted frozen droplets may be removed from the sol (e.g. using filtration) and then phase inversion carried out to precipitate the biopolymer and form beads/particles thereof.

[0204] Having generally described this disclosure, a further understanding can be obtained by reference to certain specific examples illustrated below which are provided for purposes of illustration only and are not intended to be all inclusive or limiting unless otherwise specified. Examples

Materials and methods

Preparation of cellulose solutions

[0205] Microcrystalline cellulose (MCC, from Sigma-Aldrich®) and EmimOAc were dried in a vacuum oven at 80°C for 1 h to remove traces of water. Cellulose solutions were prepared at a concentration of 8 wt% MCC in the EmimOAc with 8 wt% deionized water content. The water was first added to the EmimOAc under stirring, followed by the MCC. The mixture was shaken by hand for a minute, then transferred to rollers for 24 h. The samples were placed in a 70°C oven for 24 h, stirred with a spatula, left in the oven for a further 24 h, and then finally transferred to the rollers once again for 24 h.

[0206] Alternatively, cellulose solutions were prepared by dispersing the MCC in dimethyl sulfoxide (DMSO) with an overhead stirrer (900 rpm) at room temperature before EmimOAc was added dropwise into the dispersion and the mixture was stirred for 4 h. The solvent ratio of DMSO to EmimOAc was 70:30 w/w.

Preparation of cellulose beads by extrusion

[0207] The cellulose solution prepared as detailed above was loaded into a syringe fitted with a blunt-tipped needle. The solution was immediately extruded from the needle dropwise at 0.1 mL/min via a syringe pump (KdScientific-210) into an anti-solvent that was water or ethanol. An appropriate dropping height for optimal sphericity was selected by eye for each sample. The beads were washed with deionised water.

Preparation of cellulose beads by membrane emulsification

[0208] A dispersed phase comprising 8 wt% microcrystalline cellulose and 8 wt% water in 1- ethyl-3-methylimidazolium acetate was prepared according to routine methods known in the art. This dispersed phase had a transition temperature (e.g. freezing point) of approximately -5°C. An aqueous continuous phase was also prepared according to routine methods known in the art. The continuous phase had a pour point of -15°C.

[0209] The dispersed phase and continuous phase were fed into a membrane emulsification unit and an emulsion thereby formed. The emulsion was then cooled to a temperature between 0 and 11 °C before being transferred into a phase inversion unit with an aqueous anti-solvent to form cellulose particles. [0210] Cooling of the emulsion was carried out with an immersed coil heat exchanger. The immersed coil heat exchanger was chosen to maintain a laminar flow and minimise flow disturbances as the emulsion cooled. The coil heat exchanger contained a length (L) of coiled tubing with diameter D and pitch P, in a cold water bath at 0°C and was sufficient to cool a 0.5 L/min emulsion to below 11 °C. The temperature of the emulsion was monitored with a thermometer at the exit of the coil heat exchanger. Spherical biopolymer particles were thereby obtained.

Example 1 : Preparation of dialdehyde cellulose beads

[0211] 10 g of cellulose beads were prepared from an 8 wt% cellulose solution according to the methods detailed above. The cellulose beads (10 g) were suspended in 40 mL of sodium periodate at a concentration of 50 mM and the reaction was allowed to proceed at 20 °C for 3.5 h under mild agitation using an overhead stirrer. Subsequently, the sodium periodate was removed by separating the beads using a stainless-steel sieve and washed with deionised water until the absorption of the supernatant at 290 nm (corresponding to the absorption peak of sodium periodate) was zero. The washed beads were then stored in deionised water at room temperature. The degree of oxidation of the oxidised beads was determined to be approximately 10% (based on the number of moles of carbonyl groups per mole of anhydrous glucose units [AUG]) by the method of reaction with hydroxylamine hydrochloride and titration with sodium hydroxide as detailed herein.

Example 2: Amination of dialdehyde cellulose beads

[0212] 20 g of dialdehyde cellulose beads in water were prepared as detailed in Example 1. The beads were solvent exchanged from water to methanol by washing the beads twice with methanol. The beads (20 g) were then transferred to a 50 mL disposable centrifuge tube, to which was added 25 mL of methanol. 1.2 mole equivalents (relative to the amount of carbonyl groups present on the beads) of diamine (1 ,6-diaminohexane; 1 ,8-diaminooctane; or 1 ,10- diaminodecane) were dissolved into the methanol/beads mixture. The reaction was allowed to proceed at room temperature for 24 h, after which the beads were sieved and immersed into a solution containing 1 .2 mole equivalents (relative to the amount of amount of carbonyl groups present in the dialdehyde cellulose bead starting material) of sodium borohydride in methanol. The reacted beads were subsequently washed thoroughly with deionised water to remove all traces of the methanol and sodium borohydride. Example 3: Amination of dialdehyde cellulose beads in water

[0213] Aminated cellulose beads were prepared using 1 ,6-diaminohexane or 1 ,8- diaminooctane as detailed in Example 2, except that the steps of reaction with the diamine and subsequent reduction with sodium borohydride were each performed in deionised water. 1.5 mole equivalents of sodium borohydride were used to compensate for the reduced stability of the sodium borohydride in water.

[0214] For Examples 2 and 3, the reaction scheme can be depicted as set out below: where ‘DAC’ refers to dialdehyde cellulose, and ‘DABs’ refers to diaminated beads.

Example 4: Enzyme functionalisation of aminated cellulose beads

[0215] Equal volumes of aminated cellulose beads prepared according to Example 3, which had been stored in deionised water and not allowed to dry at any point, were immersed into lipase solutions having enzyme concentrations of 3-12 mg/mL. The resulting mixtures were incubated at room temperature for 24 hours. The lipases used were Candida antartica lipase B (CalB), Thermomyces lanuginosus lipase (TL Lipolase), and Pseudomonas cepacia lipase (Amano PS), each of which were obtained from ChiralVision B.V., Netherlands.

[0216] It was found that functionalisation with the hydrophobic diamine moieties had a marked effect on the degree of adsorption of enzyme on the beads. Enzyme adsorption was determined by measuring the amount of free enzyme remaining in solution after incubation with the aminated cellulose beads. Thus, a lower amount of free enzyme in solution after incubation indicates a higher degree of adsorption.

[0217] As can be seen in Figure 3, cellulose beads aminated with each of 1 ,6-diaminohexane; 1 ,8-diaminooctane; or 1 ,10-diaminodecane each exhibited significantly greater adsorption of enzyme compared to unmodified and dialdehyde-cellulose beads. [0218] In addition, the use of water or ethanol as the anti-solvent in the preparation of the beads prior to functionalisation did not have a significant impact on the adsorption of the enzymes (see Figure 4).

Example 5: Measurement of enzyme activity

[0219] Enzyme activity was determined in vitro by measuring the conversion over time of p- nitrophenyl butyrate to p-nitrophenol and butyric acid by UV-vis spectrophotometry. The reaction scheme for the assay is shown below: p-Nitrophenyl butyrate Water Butyric acid p-nitrophenol Hydrogen

[0220] About 0.6 mg of enzyme functionalised beads as prepared in Example 4 were suspended in 2 mL of Phosphate Saline Buffer (PBS, pH ~7.4) containing 1 mM of p- nitrophenyl butyrate. Reaction progress was monitored continuously by UV-vis spectrophotometry at 25 °C by measuring the absorbance at 400 nm for approximately 5 min. One unit (U) of activity corresponds to the release of 1 micromole of p-nitrophenol per minute at pH 7.4 and 25 °C using p-nitrophenyl butyrate as substrate. Activity was calculated according to the following formula and reported in terms of Units (U) of activity per dry weight of functionalised beads (U/g):

U _ [( 4 ₄ o _Onm / min Beads) - A _400nm / in Blank ] g(Beads) £ x I x g(Beads) x 1000 wherein:

A oonm = absorbance (final) - absorbance (initial);

E = 0.01725 |JM- ¹ cm- ¹ ;

I = 1 cm; g(Beads) = dry weight of beads in grams.

[0221] As can be seen from a comparison of Figure 4 and Figures 5a, 5b and 5c (Cycle 1), the enzyme immobilised aminated beads exhibited a higher activity than enzyme immobilized on unmodified beads. Moreover, the enzyme immobilised aminated beads showed better retention of activity upon recycling. Retention of activity was followed by performing consecutive enzyme activity measurements using recycled functionalised beads. For each cycle, the beads were washed thoroughly with PBS to remove substrate and products from the preceding activity measurement. The results of these measurements are shown in Figures 5a, 5b and 5c.

[0222] The stability of enzyme activity under storage conditions was also studied. To determine storage stability, functionalised beads were stored in deionised water at about 4 °C. At 1 , 2, 3, and 4 month intervals, samples of the functionalised beads were withdrawn from the storage vessels and enzyme activity was measured as described above. As can be seen from Figures 6a, 6b and 6c, the aminated beads showed good retention of activity for storage periods of at least 4 months.

Example 6: Morphological characterisation of functionalised beads

[0223] To determine the morphology of the functionalised beads, aminated beads in the form of a hydrogel as prepared by the methods detailed above and which had been stored in water were freeze-dried in a lyophiliser before being imaged by SEM. Micrographs were obtained using a JEOL SEM648OLV microscope. The samples were flash frozen in liquid nitrogen and lyophilized using a MiniLyotrap (LTE scientific). Cross sections were prepared by cutting with sharp blades before the flash freezing process. Prior to imaging, the samples were gold coated (Edwards sputter coater, S150B) for 5 min. Representative SEM images of whole beads, the bead surface, and the internal structure of beads aminated with either 1 ,6-diaminohexane, 1 ,8- diaminooctane, or 1 ,10-diaminodecane and upon which TL Lipolase has been immobilised are shown in Figure 7.

[0224] As can be seen, functionalisation with diamines having longer hydrocarbyl chains, which are more hydrophobic, produced functionalised beads with a smoother surface. The internal average pore size of the beads also decreased with the increasing hydrophobicity of the diamine.

[0225] The various embodiments described herein are presented only to assist in understanding and teaching the claimed features. These embodiments are provided as a representative sample of embodiments only, and are not exhaustive and/or exclusive. It is to be understood that advantages, embodiments, examples, functions, features, structures, and/or other aspects described herein are not to be considered limitations on the scope of the invention as defined by the claims or limitations on equivalents to the claims, and that other embodiments may be utilised and modifications may be made without departing from the scope of the claimed invention. Various embodiments of the invention may suitably comprise, consist of, or consist essentially of, appropriate combinations of the disclosed elements, components, features, parts, steps, means, etc., other than those specifically described herein. In addition, this disclosure may include other inventions not presently claimed, but which may be claimed in future.