Functionalised biopolymer particle preparation

Abstract

The present disclosure provides methods for preparing functionalised biopolymer particles. One aspect provides a method comprising mixing a solvent dispersion of biopolymer particles with a first compound selected from a monocarboxylic acid, a dicarboxylic acid and a cyclic anhydride, and reacting the biopolymer particles with the first compound via an esterification reaction to form the functionalised biopolymer particles. Also provided are functionalised biopolymer particles obtained from the methods as well as uses thereof.

Description

Functionalised biopolymer particle preparationFIELD

[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 methods and uses thereof.BACKGROUND

[0002] Biopolymers such as polysaccharides are an important development in the reduction of the environmental footprint of consumer and industrial products. Biopolymers and biopolymer particles such as beads 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 unplasticisedmonomers 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 retention of activity over time and e.g. during use and re-use. For industrial applicability, it is desirable for any such approach to be efficient, such as minimising the number of reaction steps, minimising the amount of consumables (e.g. organic solvents that cannot be recovered and reused), and / or avoiding the use of hazardous reagents, solvents, etc. that may be prohibited or undesirable for downstream processes and / or which would require specialist handling and equipment in the industrial setting. In turn, there remains a need for cost-effective processes suitable for large- scale industrial application and which may also provide further environmental / sustainability benefits. The present disclosure meets the foregoing needs with the various aspects and embodiments defined herein.SUMMARY

[0009] In a first aspect, the present disclosure provides a method for preparing functionalised biopolymer particles, said method comprising: mixing a solvent dispersion of biopolymer particles with a first compound, wherein the first compound comprises at least one carboxyl group or is an acid anhydride, and reacting the biopolymer particles with the first compound via an esterification reaction to form the functionalised biopolymer particles. In some embodiments the method comprises the steps of: (i) providing the dispersion of biopolymer particles; and (ii) reacting the biopolymer particles with the first compound via an esterification reaction to form the functionalised biopolymer particles.

[0010] In a second aspect, the present disclosure provides a method for immobilising enzymes on functionalised biopolymer particles, wherein the method comprises preparing functionalised particles by the method of the first aspect as described herein, and contacting the functionalised particles with an enzyme. In the second aspect, the functionalised particles may also be prepared by the method of the sixth aspect as described herein.

[0011] In a third 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.

[0012] In a fourth aspect, the present disclosure provides for the use of the functionalised biopolymer particles obtained by the methods of the first aspect as described herein for immobilising an enzyme. The functionalised particles may also be obtained by the method of the sixth aspect as described herein. Features described herein in the context of the methods and particles are applicable to the use.

[0013] In a fifth aspect, the present disclosure provides a method for performing an enzyme- catalysed reaction, the method comprising the step of forming a reaction mixture comprising functionalised biopolymer particles prepared by prepared by the method of the second aspect described herein and at least one substrate. Features described herein in the context of the preparation method, immobilisation method and particles are applicable to this method.

[0014] In a sixth aspect, the present disclosure provides a method for preparing functionalised biopolymer particles from a solvent dispersion of biopolymer particles, wherein the solvent of the dispersion is aqueous and wherein the biopolymer particles are in the form of a hydrogel, said method comprising: optionally subjecting the solvent dispersion of biopolymer particles to a solvent exchange; optionally filtering the biopolymer particles; drying the biopolymer particles to remove the solvent; mixing the dried biopolymer particles with a first compound, wherein the first compound comprises at least one carboxyl group or is an acid anhydride, and reacting the biopolymer particles with the first compound via an esterification reaction to form the functionalised biopolymer particles.Features described herein in the context of the first aspect are applicable to this method unless indicated explicitly or by the context of the sixth aspect.

[0015] In a still further aspect, the present disclosure provides for the use of functionalised biopolymer particles prepared by the method of the second aspect as described herein for catalysing a reaction. Features described herein in the context of the preparation method, the immobilisation method and obtained particles are applicable to this use.

[0016] 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.

[0017] 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

[0018] Figure 1 is a schematic representation of an embodiment wherein the biopolymer particles are prepared by extruding a dispersed phase into an anti-solvent.

[0019] 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)).

[0020] Figure 3A shows representative ATR-FTIR spectra of unmodified cellulose beads compared to itaconic anhydride (ITA) functionalised cellulose beads prepared according to Example 1 (Method A). The zoomed region of the spectra highlights the peak at about 1715 cm-1seen for the functionalised beads, which could be assigned to the carbonyl stretch (C=O) of the itaconate moieties resulting from the esterification reaction of the present disclosure.

[0021] Figure 3B shows representative FTIR spectra of unmodified cellulose beads compared to functionalised cellulose beads prepared according to Example 2 (Method B1). “NB-ITA ester 4” was functionalised with ITA in the presence of DMAP as a nucleophilic catalyst. “NB-ITA ester 5” was functionalised with ITA in the absence of DMAP. “NB-ITA ester 6” was functionalised with ITA in the absence of DMAP. The zoomed region of the spectra highlightsthe peak at about 1715 cm-1for the functionalised biopolymer particles, which could be assigned to the carbonyl stretch (C=O) of the itaconate moieties resulting from the esterification reaction of the present disclosure.

[0022] Figure 3C shows representative FTIR spectra of unmodified cellulose beads compared to functionalised cellulose beads prepared according to Example 3 (Method B2). “NB-ITA ester 7” was functionalised with ITA in the absence of DMAP, with a reaction time of 5 h at 80 °C. The zoomed region of the spectra highlights the peak at about 1715 cm-1for the functionalised biopolymer particles, which could be assigned to the carbonyl stretch (C=O) of the itaconate moieties resulting from the esterification reaction of the present disclosure.

[0023] Figure 4 shows the degree of enzyme (Thermomyces lanuginosus lipase (TLL) adsorption on unmodified cellulose beads compared to ITA-functionalised cellulose beads prepared according to Example 2 (Method B1). “NB-ITA ester 4” was functionalised with ITA in the presence of DMAP as a nucleophilic catalyst. “NB-ITA ester 6” was functionalised with ITA in the absence of DMAP. The degree of adsorption was determined by measuring the percentage enzyme adsorbed from solution and the mass fraction of enzyme loading after incubation with the beads. Error bars represent standard deviations (n = 3). Retention was determined in a similar manner after washing with phosphate-buffered saline (PBS).

[0024] Figure 5 shows the initial lipase activity as determined according to Example 6 and expressed as units per gram (ll / g) for unmodified beads and functionalised beads prepared according to Example 2 to each of which Thermomyces lanuginosus lipase (TLL) was subsequently adsorbed. “NB-ITA ester 4” was functionalised with ITA in the presence of DMAP as a nucleophilic catalyst. Error bars represent standard deviations (n = 3)DETAILED DESCRIPTION

[0025] 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.

[0026] As used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0027] 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.

[0028] 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.

[0029] As used herein, the term “at least” includes the end value of the range that is specified. For example, “at least the melting point of the first compound” includes the melting point of the first compound.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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 disclosurecontemplates 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.

[0034] “Alkyl" is defined herein as a straight-chain or branched alkyl radical, preferably C1.20 alkyl, more preferably C1-12 alkyl, even more preferably C1.10 alkyl or C1.6 alkyl, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl. Most preferably, the alkyl is a C1.3 alkyl.

[0035] “Cycloalkyl" is defined herein as a cyclic alkyl ring, preferably, C3-7-cycloalkyl, more preferably Cs-e-cycloalkyl. Preferred examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl, or a fused bicyclic ring system such as norbornane.

[0036] As used herein, the term “ary / ” or “aromatic" refers to a Ce-12 aromatic group, which may be benzocondensed, for example, phenyl or naphthyl.

[0037] “Halogen” or “halo” is defined herein as chloro, fluoro, bromo or iodo. Similarly, “halide” is defined herein as chloride, fluoride, bromide or iodide.

[0038] “Haloalkyl” is defined herein as a straight-chain or branched alkyl radical as defined above, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, that is substituted with one or more halogen atoms (that may be the same or different), such as fluorine, chlorine, bromine, and iodine. Preferably, the haloalkyl group is a C1.20 haloalkyl, more preferably C1-12 haloalkyl, even more preferably C1.10 haloalkyl or Ci-e haloalkyl. Preferred examples are C1.3 halofluorides such as CF3 and CHF2, with CF3 being particularly preferred.

[0039] “Alkoxy” is defined herein as an oxygen atom bonded to an alkyl group as defined above. Preferably the alkoxy group is a C1.20 alkoxy, more preferably C1-12 alkoxy, even more preferably C1.10 alkoxy or Ci-e alkoxy, for example methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy and hexoxy. A particularly preferred group is C1.3 alkoxy, such as methoxy (-OCH3).

[0040] “Haloalkoxy” is defined herein as an alkoxy group as described above substituted with one or more halogen atoms (that may be the same or different), such as fluorine, chlorine, bromine, and iodine. Preferably, the haloalkoxy group is a C1.20 haloalkoxy, more preferably C1-12 haloalkoxy, even more preferably C1.10 haloalkoxy or Ci-e haloalkoxy. A particularly preferred group is Ci-e haloalkoxy, such as OCF3.

[0041] “Heteroaryl” is defined herein as a monocyclic aromatic ring comprising one or more heteroatoms (that may be the same or different), such as oxygen, nitrogen or sulphur.Examples of suitable 6-membered heteroaryl groups include pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl and triazinyl.

[0042] “Aralkyl’ is defined herein as an alkyl group as defined above substituted by one or more aryl groups as defined above.

[0043] “Heterocycloalkyl" refers to a cyclic aliphatic group containing one or more heteroatoms selected from nitrogen, oxygen and sulphur, which is optionally interrupted by one or more - (CO)- groups in the ring and / or which optionally contains one or more double bonds in the ring. Preferably, the heterocycloalkyl group is monocyclic or bicyclic. Preferably, the heterocycloalkyl group is a C3-7-heterocycloalkyl, more preferably a Cs-e-heterocycloalkyl. Alternatively, the heterocycloalkyl group is a C4-7-heterocycloalkyl, more preferably a C4-6-heterocycloalkyl. Preferred heterocycloalkyl groups include, but are not limited to, piperazinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, oxetanyl and azetidinyl. Preferably, the heterocycloalkyl group is monovalent.

[0044] As used herein, the term “alkenyl" refers to both straight and branched carbon chains which have at least one carbon-carbon double bond. In some embodiments, alkenyl groups may include C2-C12 alkenyl groups. In other embodiments, alkenyl includes C2-C10, C2-C8, C2- Ce or C2-C4 alkenyl groups. In one embodiment of alkenyl, the number of double bonds is 1-3; in another embodiment of alkenyl, the number of double bonds is one. Other ranges of carboncarbon double bonds and carbon numbers are also contemplated depending on the location of the alkenyl moiety on the molecule. “C2-Cio-alkenyl” groups may, for example, include more than one double bond in the chain.

[0045] As used herein, the term “alkynyl" refers to both straight and branched carbon chains which have at least one carbon-carbon triple bond. In some embodiments, alkynyl groups may include C2-C12 alkynyl groups. In other embodiments, alkynyl includes C2-C10, C2-C8, C2-C6 or C2-C4 alkynyl groups. In one embodiment of alkynyl, the number of triple bonds is 1-3; in another embodiment of alkenyl, the number of triple bonds is one. A particularly preferred alkynyl group is -C=CH.

[0046] As used herein, the term “alkylene" refers to a linear or branched saturated divalent hydrocarbon radical. Preferably, the alkylene group is a linear saturated divalent hydrocarbon radical comprising one to six carbon atoms, or a branched saturated divalent hydrocarbon radical comprising three to six carbon atoms.

[0047] 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-cyclopentyl propyl, 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, heterocycloalkyl, aralkyl or alkenyl group.

[0048] As used herein, preferably amino is -NR2 wherein each R group is selected from hydrogen, alkyl, cycloalkyl, and aryl groups, preferably wherein at least one R group is selected from hydrogen, alkyl, cycloalkyl, and aryl groups; in which each alkyl, cycloalkyl and aryl group is optionally substituted.

[0049] As used herein, preferably alkyl is Ci-Ce alkyl, haloalkyl is Ci-Ce haloalkyl, haloalkoxy is Ci.Ce haloalkoxy, and alkoxy is Ci.Ce alkoxy.

[0050] 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 a first compound as described herein, 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.

[0051] Thus, in a first aspect, the present disclosure provides a method for preparing functionalised biopolymer particles, said method comprising mixing a solvent dispersion of biopolymer particles with a first compound, wherein the first compound comprises at least onecarboxyl group or is an acid anhydride, and reacting the biopolymer particles with the first compound via an esterification reaction to form the functionalised biopolymer particles.

[0052] In the present disclosure, it has been found that functionalisation of the biopolymer particles with the first compound as defined herein via an esterification reaction according to the present disclosure confers advantageous physicochemical properties over unmodified biopolymer particles. In particular, said functionalisation has been found to enhance the degree of adsorption of enzymes by the biopolymer particles. Moreover, the functionalised biopolymer particles of the present disclosure upon which enzymes have been immobilised exhibit good retention of adsorbed enzyme. The methods described herein may provide additional benefits including those relating to cost-efficiency, ease of industrial scale-up, and environmental / sustainability. For example, the methods described herein are advantageous over prior art methods in that a single reaction step for functionalisation via the esterification reaction is required, thereby providing a simplified process. Further, the first compound as described in more detail herein may advantageously be derived by biotechnological processes rather than from petrochemicals, for example from biomass. The use of bio-derived reagents rather than those derived from e.g. petrochemicals may be advantageous in reducing the environmental impact of the method of the present disclosure, particular with respect to existing methods in the prior art. Moreover, in various embodiments the use and / or consumption of solvents such as organic solvents is minimised. The methods of the present disclosure also minimise or avoid the need to use hazardous reagents, such as those that may be difficult and / or expensive to use for industrial scale-up. Additionally, the methods of the present disclosure may enable the recovery and reuse of one or more reaction components not consumed during the reaction. The functionalised biopolymer particles may also exhibit good mechanical stability during preparation and their subsequent use, including during storage and recycling.

[0053] 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.

[0054] 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 themonomeric 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.

[0055] 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, cellulose, starch, agarose, dextran, chitosan, pectin or glycogen. Most preferably the biopolymer is cellulose. In other words, in said preferred embodiment the biopolymer particles are cellulose particles.

[0056] 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. The cellulose may, for example, be virgin, recycled, pulp and / or microcrystalline cellulose. In various embodiments, cellulose is 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. Recycled sources may provide an additional environmental benefit to the method of the present disclosure.

[0057] In various embodiments the cellulose is microcrystalline cellulose (MCC); 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.

[0058] In various embodiments of the present disclosure, the cellulose is regenerated cellulose. Regenerated cellulose is produced by the conversion of natural cellulose (e.g. from wood or plant fibers) to a soluble cellulosic derivative and subsequent regeneration (e.g. by contact with an anti-solvent). Regenerated cellulose is commercially available and the selection of suitable regenerated celluloses is within the common general knowledge of a person of skill in the art of the present disclosure. Suitable materials include Rayon and Lyocell.

[0059] The term “particle” used herein 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 theparticles 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.

[0060] In various embodiments of the present disclosure, the biopolymer particles are in the form of beads. The term “bead” as used herein refers to a discrete entity with defined size and shape that does not have any dimension or dimensions substantially greater than any other dimension and which may be described as “spherical” or “substantially spherical”. A person skilled in the art is readily able to identify a “bead” as opposed to e.g. an elongate particle. In other words, the term “bead” as used herein does not include elongate particles such as (nano / micro)fibres, (nano / micro)fibrils and the like, nor does said term include flat / sheet-like structures such as membranes and the like. As used herein, embodiments referring to “beads” as such apply equally to each of the aspects of the present disclosure unless specifically indicated and include any biopolymer bead as encompassed by the present disclosure.

[0061] 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.

[0062] 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 or beads may have a diameter of at least about 1 pm. In some embodiments, the particles or beads may have a diameter of at least about 10 pm. In some embodiments, the particles or beads may have a diameter of at least about 25 pm. In some embodiments, the particles or beads may have a diameter of at least about 50 pm. In some embodiments, the particles or beads may have a diameter of at least about 100 pm.

[0063] In some embodiments, the particles or beads may have a diameter of less than about 5 mm. In some embodiments, the particles or beads may have a diameter of less than about4 mm. In some embodiments, the particles or beads may have a diameter of less than about3 mm. In some embodiments, the particles or beads may have a diameter of less than about2 mm. In some embodiments, the particles or beads may have a diameter of less than about1 mm.

[0064] In various embodiments, the biopolymer particles have a diameter of from about 1 pm to about 3 mm. In various embodiments, the biopolymer particles have a diameter of from about 10 pm to about 3 mm. In various embodiments, the biopolymer particles have a diameter of from about 25 pm to about 3 mm. In various embodiments, the biopolymer particles have a diameter of from about 50 pm to about 3 mm. In the foregoing embodiments, the biopolymer particles are preferably approximately spherical.

[0065] In various embodiments, the biopolymer particles have a diameter of from about 0.1 mm to about 3 mm. In various embodiments, the biopolymer particles have a diameter of from about 0.15 mm to about 3 mm. In various embodiments, the biopolymer particles have a diameter of about 0.20 mm to about 3 mm. In the foregoing embodiments, the biopolymer particles are preferably approximately spherical.

[0066] In various embodiments, the biopolymer particles have a diameter of from about 1 pm to about 2 mm. In various embodiments, the biopolymer particles have a diameter of from about 10 pm to about 2 mm. In various embodiments, the biopolymer particles have a diameter of from about 25 pm to about 2 mm. In various embodiments, the biopolymer particles have a diameter of from about 50 pm to about 2 mm. In the foregoing embodiments, the biopolymer particles are preferably approximately spherical.

[0067] In various embodiments, the biopolymer particles have a diameter of from about 1 pm to about 1 mm. In the foregoing embodiments, the biopolymer particles are preferably approximately spherical. The biopolymer particles in such embodiments may otherwise be referred to as “microspheres”.

[0068] 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. In the foregoing embodiments, the biopolymer particles are preferably approximately spherical.

[0069] Biopolymer particles may be obtained from various methods of production, including those specifically described herein, in a form wherein said particles are wetted or immersed in a solvent such as water. Such particles may be referred to as “wet” and may be provided in this form for further use. In some embodiments, the biopolymer particles are in the form of a hydrogel as defined herein. Biopolymer particles in the form of a hydrogel may, in various embodiments, be obtained by the processes further described herein below. It has been found that such biopolymer particles in the form of a hydrogel are particularly advantageous whenused in the esterification reaction of the present disclosure, such as in terms of the efficiency and / or degree of functionalisation, which may in turn be associated with advantages where used e.g. for enzyme immobilisation. In various embodiments of the methods described herein, the biopolymer particles preferably have not been previously dried before carrying out the esterification reaction. In other embodiments of the methods described herein, the biopolymer particles, which may be in the form of a hydrogel, may be dried before carrying out the esterification reaction.

[0070] Thus, the method of the first aspect and of the sixth aspect comprises a solvent dispersion of biopolymer particles; this may otherwise be referred to herein as a dispersion of biopolymer particles in a solvent. As used herein, the term “dispersion” includes the biopolymer particles being wetted or immersed in the solvent. Thus, the solvent may be any substance (e.g. a liquid) in which the biopolymer particles may be dispersed. The term “solvent” also includes solvent mixtures. The biopolymer particles may be evenly or unevenly dispersed in the solvent. In various embodiments the biopolymer particles in the solvent may be manipulated e.g. by mixing or other forms of agitation, for example to provide an even dispersion and / or to maximise contact and thereby reaction with other substances such as the first compound of the method. A person of skill in the art will be able to select suitable means of providing manipulation, which may include paddle mixers, magnetic stirrer bars etc.

[0071] The solvent in which the dispersion of biopolymer particles is provided is not limited and may include any solvent used in the preparation of the biopolymer particles. Thus, the embodiments of the solvent disclosed herein in relation to the preparation of biopolymer particles may be combined with any of the embodiments of the first aspect and sixth aspect disclosed herein. For example, the solvent may be an organic solvent, an aqueous solvent, or an inorganic nonaqueous solvent, or a combination thereof. Preferably the solvent of the dispersion is aqueous. As used herein, an “aqueous solvent” refers to water that may comprise at least one further substance, for example ions and / or one or more additional solvents such as an organic solvent or an inorganic nonaqueous solvent (e.g. as a co-solvent). The term “aqueous” being understood, as is common general knowledge in the art, as “water” being the solvent.

[0072] In various embodiments, the solvent in which the dispersion of biopolymer particles is provided may therefore be an aqueous solvent and thereby comprise 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 may be aqueous and thereby comprise water and one or more ionic liquid(s). In various embodiments of the presentdisclosure, the solvent in which the dispersion of biopolymer particles is provided may consist essentially of water.

[0073] Non-limiting examples of solvents 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, tetra hydrofuran, 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 identification of suitable solvents for the dispersion of the present disclosure is within the common general knowledge of the skilled person.

[0074] As noted herein above, the solvent in which the dispersion of biopolymers is provided may be the same solvent in which the biopolymer particles were prepared. In such embodiments the solvent is preferably aqueous; the term “aqueous” being as defined above. In various embodiments of the disclosure, the biopolymer particles are in the form of a hydrogel. A hydrogel is defined by the International Union of Pure and Applied Chemistry (IUPAC) 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.

[0075] In various embodiments, the solvent of the dispersion is aqueous, and the biopolymer particles are in the form of a hydrogel. In various embodiments, the method may further comprise subjecting the biopolymer particles to a solvent exchange prior to the esterification reaction. As used herein, “solvent exchange” means that the solvent in which the biopolymer particles are dispersed is exchanged, for example from an aqueous solvent to an organic solvent. Thus, in various embodiments of the present disclosure, the solvent of the dispersion is aqueous, and the method further comprises subjecting the biopolymer particles to a solvent exchange with an organic solvent prior to the esterification reaction. In such embodiments, the biopolymer particles are preferably in the form of a hydrogel.

[0076] The solvent exchange may be effected by any suitable method that will be commonly known to a person of skill in the art, for example by vacuum filtration. For instance, the dispersion of biopolymer particles in a solvent, e.g. an aqueous solvent, may be subjected to at least one cycle of vacuum filtration to substantially remove the solvent from the biopolymer particles followed by re-dispersion of the biopolymer particles in a different solvent, e.g. an organic solvent when the biopolymer particles are dispersed in an aqueous solvent. For instance, the biopolymer particles may be subjected to at least 1 , at least 2, or at least 3 solvent exchange cycles, such as from an aqueous solvent to an organic solvent and vice versa.

[0077] In various embodiments, the solvent exchange comprises an organic solvent and this organic solvent may be selected from acetone, acetonitrile, benzene, 1-butanol, 2-butanol, 2- butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1 ,2- dichloroethane, diethylene glycol, diethyl ether, diethylene glycol dimethyl ether, 1 ,2-dimethoxy ethane (DME), dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), 1 ,4-dixoane, ethanol, ethyl acetate, ethylene glycol, glycerol, heptane, hexamethylphosphoramide (HMPA), hexamethylphosphorous triamide (HMPT), hexane, methanol, methyl t-butyl ether (MTBE), methylene chloride, N-methyl-2-pyrrolidinone (NMP), nitromethane, pentane, petroleum ether, 1-propanol, 2-propanol, pyridine, tetra hydrofuran (THF), toluene triethyl amine, xylene, dimethylacetamide (DMAc), and mixtures thereof.

[0078] In various embodiments, the organic solvent used for the solvent exchange is preferably anhydrous. As used herein, the term “anhydrous” means substantially free of water. For example, the anhydrous solvent may comprise less than 100 ppm water, less than 10 ppm water or less than 1 ppm water. Anhydrous solvents are commercially available and also readily obtained by methods commonly known in the art such via drying trains, solvent stills, and / or the use of desiccants and drying agents such as molecular sieves. Water content is also readily determined by methods commonly known in the art such as Karl Fischer titration.

[0079] In other embodiments, the biopolymer particles are dried prior to the esterification reaction.

[0080] In various embodiments, the drying may be performed after a solvent exchange step and / or a filtration step such as those described herein above. For example, in various embodiments the biopolymer particles are provided in the form of a hydrogel, subjected to a solvent exchange step and / or a filtration step and then dried prior to the esterification reaction.

[0081] In various embodiments, drying is carried out to remove substantially all solvent from the biopolymer particles. It will be understood that where the biopolymer particles have been subjected to a solvent exchange, e.g. into an organic solvent, the solvent removed will bepredominantly the solvent into which the biopolymer particles were exchanged, e.g. the organic solvent, but may also include (where present) residual amounts of the solvent from which said particles were exchanged, e.g. the aqueous solvent. In various embodiments, the biopolymer particles are dried at room temperature and / or at a temperature of from about 25 °C to about 90 °C, from about 35 °C to about 90 °C, or from about 45 °C to about 90 °C.

[0082] In various embodiments, drying involves one or more drying steps. For example, drying may comprise drying at a first temperature (e.g. room temperature) and drying at a second temperature, wherein the second temperature is higher than the first temperature by at least about 5°C, preferably by at least about 10°C. In various embodiments, the biopolymer particles are dried at room temperature and dried at a temperature of from about 25 °C to about 90 °C, from about 35 °C to about 90 °C, or from about 45 °C to about 90 °C.

[0083] In various embodiments, drying may comprise drying at a first temperature for a first period of time and drying at a second temperature for a second period of time, wherein the second temperature is higher than the first temperature by at least about 5°C, preferably by at least about 10°C. In various embodiments, the biopolymer particles are dried at room temperature for at least 2, 4, 6, 8, 10, 12, 14 or 16 hours. In various embodiments, the biopolymer particles are dried at a temperature of from about 25 °C to about 90 °C, from about 35 °C to about 90 °C, or from about 45 °C to about 90 °C for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22 hours. Thus, in further embodiments, the biopolymer particles are dried at room temperature for at least 2, 4, 6, 8, 10, 12, 14 or 16 hours and dried at a temperature of from about 25 °C to about 90 °C, from about 35 °C to about 90 °C, or from about 45 °C to about 90 °C for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22 hours. In various embodiments, the biopolymer particles may be dried, e.g. at the temperatures and / or times set out in the foregoing embodiments, under reduced pressure. As used herein, the term “reduced pressure” means a pressure less than 1 atmosphere. In further embodiments, the biopolymer particles may be dried under vacuum. In various embodiments, the biopolymer particles may be agitated during drying.

[0084] The means by which drying is conducted are not limited and would be known to a person skilled in the art. In various embodiments, the drying at the second temperature (e.g. for a second period of time as described herein above) is carried out using oven drying.

[0085] In other embodiments wherein the biopolymer particles are dried prior to the esterification reaction, the solvent exchange may be omitted. For example, in various embodiments the biopolymer particles are provided as a solvent dispersion, wherein the solvent of the dispersion is aqueous and the biopolymer particles are in the form of a hydrogel,and wherein said biopolymer particles are dried to remove the aqueous solvent. Suitable drying methods for removing the aqueous solvent are commonly known in the art and non-limiting examples include spray drying, microwave drying, fluidised-bed drying and freeze drying. In further embodiments, the biopolymer particles may be filtered prior to drying. The skilled person is able to select suitable parameters for such methods, e.g. drying temperature, as part of their common general knowledge. In various embodiments wherein the biopolymer particles are dried to remove the aqueous solvent, the beads may be dried at a temperature of at least about 100 °C. For example, the biopolymer particles may in various embodiments by dried by spray drying, wherein the spray drying comprises a drying temperature of at least about 100 °C.Esterification

[0086] The method of the first aspect and of the sixth aspect comprises reacting the biopolymer particles with a first compound as defined herein via an esterification reaction to form the functionalised biopolymer particles. In various embodiments the first compound comprises at least one carboxyl group or is an acid anhydride. For instance, the first compound may be a monocarboxylic acid, a dicarboxylic acid or an acid anhydride. In various embodiments, the acid anhydride is preferably a cyclic anhydride. Thus, in various embodiments the first compound is selected from a monocarboxylic acid, a dicarboxylic acid and a cyclic anhydride. The biopolymer particles may be according to any of the embodiments described herein above.

[0087] As used herein, it will be understood that the term “acid anhydride” refers to an organic acid anhydride. As used herein, the term “organic” means a carbon-containing compound, such as a compound comprising at least one carbon-hydrogen bond and / or carbon-carbon bond.

[0088] Esterification reactions are known. In the esterification reaction of the present disclosure, the first compound as described herein generally becomes covalently bonded to the biopolymer particles, in particular via an ester bond, i.e. RC(O)O-, wherein the dash indicates a bond to a biopolymer particle and R represents a residue of the first compound, e.g. an optionally substituted hydrocarbyl group. The esterification reaction is not necessarily limited to any actual or proposed mechanism and includes any reaction of the starting materials as described herein that results in the formation of an ester bond to a biopolymer particle of the present disclosure.

[0089] In various embodiments of the present disclosure, the first compound may be optionally substituted. In various embodiments, the substituents may include one or more of hydroxyl,alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, halo, alkoxy, carboxy, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, haloalkoxy, amino, alkylamino, dialkylamino, cyano, nitro and azide. In a preferred embodiment, the optional substituents are selected from hydroxyl, Ci-e alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, halo, Ci-e alkoxy, carboxy, Ci-e alkoxycarbonyl, aryloxycarbonyl, halo-Ci-6 alkyl, halo-Ci-6 alkoxy, amino, Ci-e alkylamino, Ci-e dialkylamino, cyano, nitro, azide, and combinations thereof.

[0090] In the present disclosure, when the first compound comprises at least one carboxyl group, e.g. when the first compound is a monocarboxylic acid or dicarboxylic acid, a carboxyl group of the first compound is able to react (directly or indirectly) with a hydroxyl group in or on the biopolymer particles via an esterification reaction so as to form functionalised biopolymer particles. Similarly, the anhydride (e.g. cyclic anhydride) is able to react in a similar manner with hydroxyl groups in or on the biopolymer particles via an esterification reaction so as to form functionalised biopolymer particles. Accordingly, the first compound is not necessarily limited other than the presence of said functional groups.

[0091] In various embodiments the first compound is saturated. As used herein “saturated” refers to carbon-carbon bonding and does not include bonds involving heteroatoms (e.g. carbonyl groups). Non-limiting examples of saturated monocarboxylic acids include propionic acid, butanoic acid (butyric acid), pentanoic acid (valeric acid), hexanoic acid (caproic acid), heptanoic acid (enanthic acid), cyclohexanecarboxylic acid, octanoic acid (caprylic acid), nonanoic acid (pelargonic acid), decanoic acid (capric acid), undecanoic acid, dodecanoic acid (lauric acid), tridecanoic acid, tetradecanoic acid (myristic acid), pentadecanoic acid, hexadecanoic acid (palmitic acid), heptadecanoic acid, octadecanoic acid (stearic acid), nonadecanoic acid, icosanoic acid (arachidic), heneicosanoic acid, docosanoic acid (behenic acid), tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid and isomeric forms thereof.

[0092] Non-limiting examples of saturated dicarboxylic acids include malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, 1 ,4-cyclohexanedicarboxylic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, brassylic acid, thapsic acid, japanic acid, phellogenic acid and isomeric forms thereof.

[0093] Non-limiting examples of saturated cyclic anhydrides include succinic anhydride, methylsuccinic anhydride, glutaric anhydride, and adipic anhydride.

[0094] In various embodiments, the first compound is preferably unsaturated. As used herein “unsaturated” refers to carbon-carbon bonding and does not include bonds involving heteroatoms (e.g. carbonyl groups). In various embodiments, the unsaturated first compoundis preferably aliphatic. In various embodiments the first compound comprises an alkene group. In various embodiments, the first compound comprises a terminal alkene group. Non-limiting examples of unsaturated monocarboxylic acids include acrylic acid, propargylic acid, crotonic acid, isocrotonic acid, methacrylic acid, vinylacetic acid, sorbic acid, benzoic acid, 2-octenoic acid, cinnamic acid, oleic acid, linoleic acid, linolenic acid, stearidonic acid, arachidonic acid, EPA ((5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11 ,14-pentaenoic acid), DHA ((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoic acid), and isomeric forms thereof.

[0095] Non-limiting examples of unsaturated dicarboxylic acids include maleic acid, fumaric acid, glutaconic acid, traumatic acid, muconic acid, citraconic acid, mesaconic acid, and itaconic acid. Non-limiting examples of unsaturated cyclic anhydrides include octenylsuccinic anhydride, maleic anhydride, itaonic anhydride, citraconic anhydride, dimethylmaleic anhydride, and isomeric forms thereof.

[0096] In various embodiments, the monocarboxylic acid may be a fatty acid. Non-limiting examples of fatty acids include caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, a-linolenic acid, stearidonic acid, eicosapentaenoic acid, cervonic acid, linoleic acid, linolelaidic acid, y-linolenic acid, dihomo-y-linolenic acid, arachidonic acid, docosatetraenoic acid, palmitoleic acid, vaccenic acid, paullinic acid, oleic acid, elaidic acid, gondoic acid, erucic acid, nerovnic acid, and Mead acid.

[0097] In various embodiments, the first compound is a C3-C26 compound, preferably a C3-C26 unsaturated compound. Thus in various embodiments the first compound is selected from a C3-C26 monocarboxylic acid, a C3-C26 dicarboxylic acid, and a C4-C26 cyclic anhydride. Preferably the first compound is selected from a C3-C26 unsaturated monocarboxylic acid, a C3-C26 unsaturated dicarboxylic acid and a C4-C26 unsaturated cyclic anhydride. In any of the foregoing embodiments, the monocarboxylic acid may be a C3-C26 fatty acid.

[0098] In various embodiments, the monocarboxylic acid may be a C4-C24, C4-C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 monocarboxylic acid. In any of the foregoing embodiments, the monocarboxylic acid may be an unsaturated monocarboxylic acid. For example, the unsaturated monocarboxylic acid may be a C4-C24, C4-C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 unsaturated monocarboxylic acid comprising an alkene group, preferably a terminal alkene group.

[0099] In various embodiments, the dicarboxylic acid may be a C4-C24, C4-C22, a C4-C20, C4- C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 dicarboxylic acid. In any of the foregoingembodiments, the dicarboxylic acid may be an unsaturated dicarboxylic acid, e.g. an unsaturated C4-C24, C4-C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4_Ci2, C4-C10, C4-C8, or C4-C6 dicarboxylic acid. For example, the unsaturated dicarboxylic acid may be a C4-C24, C4-C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-Ce dicarboxylic acid comprising an alkene group, preferably a terminal alkene group.

[0100] In various embodiments, the cyclic anhydride may be a C4-C24, C4-C22, a C4-C20, C4- C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride. In any of the foregoing embodiments, the cyclic anhydride may be an unsaturated anhydride, e.g. a C4-C24, C4-C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 unsaturated cyclic anhydride. For example, the unsaturated cyclic anhydride may be a C4-C24, C4-C22, a C4-C20, C4-C18, C4- Cie, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride comprising an alkene group, preferably a terminal alkene group.

[0101] In various embodiments, the first compound is selected from a C3-C26 monocarboxylic acid, a C3-C26 dicarboxylic acid and a C4-C26, C4-C24, C4-C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride.

[0102] In various embodiments, the first compound is a C3-C24 compound. For instance, in various embodiments the first compound is selected from a C3-C24 monocarboxylic acid, a C3- C24 dicarboxylic acid and a C4-C24 cyclic anhydride. In any of the foregoing embodiments, the monocarboxylic acid may be a C3-C24 fatty acid.

[0103] In various embodiments, the first compound is selected from a C3-C24 monocarboxylic acid, a C3-C24 dicarboxylic acid and a C4-C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride.

[0104] In various embodiments, the first compound is a C3-C22 compound. For instance, in various embodiments the first compound is selected from a C3-C22 monocarboxylic acid, a C3- C22 dicarboxylic acid and a C4-C22 cyclic anhydride. In any of the foregoing embodiments, the monocarboxylic acid may be a C3-C22 fatty acid.

[0105] In various embodiments, the first compound is selected from a C3-C22 monocarboxylic acid, a C3-C22 dicarboxylic acid and a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride.

[0106] In various embodiments, the first compound is a C3-C20 compound. For instance, in various embodiments the first compound is selected from a C3-C20 monocarboxylic acid, a C3-C20 dicarboxylic acid and a C4-C20 cyclic anhydride. In any of the foregoing embodiments, the monocarboxylic acid may be a C3-C20 fatty acid.

[0107] In various embodiments, the first compound is selected from a C3-C20 monocarboxylic acid, a C3-C20 dicarboxylic acid and a C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride.

[0108] In various embodiments, the first compound is a C3-C18 compound. For instance, in various embodiments the first compound is selected from a C3-C18 monocarboxylic acid, a C3- C18 dicarboxylic acid and a C4-C18 cyclic anhydride. In any of the foregoing embodiments, the monocarboxylic acid may be a C3-C18 fatty acid.

[0109] In various embodiments, the first compound is selected from a C3-C18 monocarboxylic acid, a C3-C18 dicarboxylic acid and a C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride.

[0110] In various embodiments, the first compound is selected from a C4-C26 monocarboxylic acid, a C4-C26 dicarboxylic acid and a C4-C24, C4-C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride.

[0111] In various embodiments, the first compound is a C4-C24 compound. For instance, in various embodiments the first compound is selected from a C4-C24 monocarboxylic acid, a C4- C24 dicarboxylic acid and a C4-C24 cyclic anhydride. In any of the foregoing embodiments, the monocarboxylic acid may be a C4-C24 fatty acid.

[0112] In various embodiments, the first compound is selected from a C4-C24 monocarboxylic acid, a C4-C24 dicarboxylic acid and a C4-C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride.

[0113] In various embodiments, the first compound is a C4-C22 compound. For instance, in various embodiments the first compound is selected from a C4-C22 monocarboxylic acid, a C4- C22 dicarboxylic acid and a C4-C22 cyclic anhydride. In any of the foregoing embodiments, the monocarboxylic acid may be a C4-C22 fatty acid.

[0114] In various embodiments, the first compound is selected from a C4-C22 monocarboxylic acid, a C4-C22 dicarboxylic acid and a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride.

[0115] In various embodiments, the first compound is a C4-C20 compound. For instance, in various embodiments the first compound is selected from a C4-C20 monocarboxylic acid, a C4-C20 dicarboxylic acid and a C4-C20 cyclic anhydride. In any of the foregoing embodiments, the monocarboxylic acid may be a C4-C20 fatty acid.

[0116] In various embodiments, the first compound is selected from a C4-C20 monocarboxylic acid, a C4-C20 dicarboxylic acid and a C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride.

[0117] In various embodiments, the first compound is a C4-C18 compound. For instance, in various embodiments the first compound is selected from a C3-C18 monocarboxylic acid, a C4- C18 dicarboxylic acid and a C4-C18 cyclic anhydride. In any of the foregoing embodiments, the monocarboxylic acid may be a C4-C18 fatty acid.

[0118] In various embodiments, the first compound is selected from a C4-C18 monocarboxylic acid, a C4-C18 dicarboxylic acid and a C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride.

[0119] In any of the foregoing embodiments, the first compound may be unsaturated. For example, in various embodiments the first compound preferably comprises an alkene group, more preferably a terminal alkene group.

[0120] In various embodiments, the first compound is a dicarboxylic acid or a cyclic anhydride. In various embodiments, the first compound is selected from a C3-C26 dicarboxylic acid and a C4-C26, C4-C24, C4-C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride. In various embodiments, the first compound is selected from a C4-C22, a C4-C20, C4- C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride. In various embodiments, the first compound is selected from a C3-C22 dicarboxylic acid and a C4-C20, C4-C18, C4-C16, C4- C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride. In various embodiments, the first compound is selected from a C3-C20 dicarboxylic acid and a C4-C18, C4-C16, C4-C14, C4-C12, C4- C10, C4-C8, or C4-C6 cyclic anhydride. In various embodiments, the first compound is selected from a C3-C18 dicarboxylic acid and a C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride.

[0121] In various embodiments, the first compound is selected from a C4-C26 dicarboxylic acid and a C4-C26, C4-C24, C4-C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride. In various embodiments, the first compound is selected from a C4-C24 dicarboxylic acid and a C4-C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4- Ce cyclic anhydride. In various embodiments, the first compound is selected from a C4-C22 dicarboxylic acid and a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride. In various embodiments, the first compound is selected from a C4-C20 dicarboxylicacid and a C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride. In various embodiments, the first compound is selected from a C4-C18 dicarboxylic acid and a C4-C16, C4- C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride.

[0122] In any of the foregoing embodiments, the first compound may be unsaturated. For example, in various embodiments the first compound preferably comprises an alkene group, more preferably a terminal alkene group. For example, in various embodiments the first compound may be an unsaturated dicarboxylic acid or an unsaturated acid anhydride, wherein the first compound comprises an alkene group, preferably a terminal alkene group. In various embodiments, the first compound is an unsaturated C4-C24, C4-C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 dicarboxylic acid or cyclic anhydride, wherein the first compound comprises an alkene group, preferably a terminal alkene group.

[0123] In various embodiments, the first compound is selected from itaconic acid, itaconic anhydride, and mixtures thereof. In various embodiments, the first compound is itaconic acid or itaconic anhydride.

[0124] In various embodiments, the first compound is a cyclic anhydride such as a C4-C24, C4- C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride. In any of the foregoing embodiments, the cyclic anhydride may be an unsaturated anhydride, e.g. a C4-C24, C4-C22, a C4-C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 unsaturated cyclic anhydride. For example, the unsaturated cyclic anhydride may be a C4-C24, C4-C22, a C4- C20, C4-C18, C4-C16, C4-C14, C4-C12, C4-C10, C4-C8, or C4-C6 cyclic anhydride comprising an alkene group, preferably a terminal alkene group.

[0125] In various embodiments, the first compound is itaconic anhydride.

[0126] In various embodiments, the first compound may advantageously be derived by biotechnological processes (rather than from petrochemicals), such as from biomass, and in particularly lignocellulosic biomass such as energy crops, agricultural and forest management residues (e.g. bagasse, woodchip), and municipal waste. In particular, lignocellulosic biomass is an important source of organic acids. Bio-derived organic acids are particularly advantageous as they are sustainable, cost-effective and environmentally friendly. For example, itaconic acid is generally industrially produced by the fermentation of glucose, molasses, or other carbohydrates by microorganisms such as fungi (e.g. Aspergillus itaconicus, Aspergillus terreus, or Ustilago maydis). In such embodiments, itaconic anhydride may be obtained from by dehydration of itaconic acid by methods known in the art, for example at temperatures of 165-180 °C and pressures of 10-30 mmHg in the presence of catalytic quantities of strong acids, such as concentrated sulphuric acid. The use of bio-derivedreagents rather than those derived from e.g. petrochemicals may be advantageous in reducing the environmental impact of the method of the present disclosure, particular with respect to existing methods in the prior art.

[0127] In various embodiments of the present disclosure, the esterification reaction may be performed in the presence of a catalyst. For example, in various embodiments the method may further comprise adding a catalyst, preferably a nucleophilic catalyst, to the reaction mixture. Thus, in various embodiments, the reaction mixture further comprises a nucleophilic catalyst. As used herein, the term “nucleophilic catalyst” includes any nucleophile that is capable of catalysing the esterification reaction of the present disclosure. A person of skill in the art of the present disclosure will understand that nucleophilic reagents are Lewis bases. The nucleophilic catalyst is not necessarily limited. The person of skill in the art will also be able to select suitable nucleophilic catalysts for esterification reactions as part of their common general knowledge.

[0128] In various embodiments, the nucleophilic catalyst is an amine compound, a N- heterocycle (preferably a N-heteroarene) or an inorganic hydroxide salt. In various embodiments, the amine compound is an acyclic or cyclic aliphatic amine or an aromatic amine. In various embodiments, the amine compound is preferably a trialkylamine or an aminosubstituted N-heteroarene. The alkyl groups of the trialkylamine may be as defined above. In various embodiments, the alkyl groups of the trialkylamine are preferably independently Ci-Ce alkyl, more preferably C1-C3 alkyl. In various embodiments, the amino substituent of the aminosubstituted N-heteroarene may be a mono- or di-alkyl amino substituent. In such embodiments, the alkyl substituents may be as defined above, preferably independently Ci-Ce or C1-C3 alkyl substituents. In various embodiments, the amine compound may be triethylamine or 4- dimethylaminopyridine (DMAP). In any of the preceding embodiments, the amine compound may be optionally further substituted. In various embodiments, the N-heterocycle is a N- heteroarene. In various embodiments the N-heteroarene is preferably a six-membered N- heteroarene, such as but not limited to pyridine, which may be optionally substituted.

[0129] In various embodiments, the inorganic hydroxide salt is an alkaline hydroxide salt, for example lithium hydroxide, potassium hydroxide or sodium hydroxide.

[0130] In various embodiments the nucleophilic catalyst is an amine compound, an N- heterocycle or an inorganic hydroxide salt, preferably an amine compound or an alkaline hydroxide salt selected from lithium hydroxide, potassium hydroxide and sodium hydroxide. In various embodiments, the nucleophilic catalyst is an acyclic or cyclic aliphatic amine, an aromatic amine, a six-membered N-heteroarene or an alkaline hydroxide salt such as lithium hydroxide, potassium hydroxide or sodium hydroxide. In various embodiments, thenucleophilic catalyst is a trialkylamine, an amino-substituted N-heteroarene, or an alkaline hydroxide salt such as lithium hydroxide, potassium hydroxide or sodium hydroxide. In various embodiments, the nucleophilic catalyst is a Ci-Ce or C1-C3 trialkylamine, an amino-substituted N-heteroarene, or an alkaline hydroxide salt such as lithium hydroxide, potassium hydroxide or sodium hydroxide. In various embodiments, the nucleophilic catalyst is a Ci-Ce or C1-C3 trialkylamine, an amino-substituted N-heteroarene, or an alkaline hydroxide salt such as lithium hydroxide, potassium hydroxide or sodium hydroxide. In various embodiments, the nucleophilic catalyst is a Ci-Ce or C1-C3 trialkylamine, a Ci-Ce or C1-C3 dialkylamino-substituted N-heteroarene, or an alkaline hydroxide salt such as lithium hydroxide, potassium hydroxide or sodium hydroxide. In any of the preceding embodiments, the amine compound may be optionally further substituted. In various embodiments, the nucleophilic catalyst may be triethylamine, 4-dimethylaminopyridine (DMAP) or an alkaline hydroxide salt such as lithium hydroxide, potassium hydroxide or sodium hydroxide.

[0131] The molar ratio of the catalyst to biopolymer particles is not necessarily limited. In various embodiments, the biopolymer is a polysaccharide and the molar ratio of the catalyst to anhydrous glucose units (AGU) of the biopolymer particles is from about 1 :100 to about 1 :1 , or from about 1 :50 to about 1 :1. In various embodiments, the biopolymer is a polysaccharide and the molar ratio of the catalyst to anhydrous glucose units (AGU) of the biopolymer particles is from about 1 :50 to about 1 :2, or from about 1 :50 to about 1 :3. For example, in various embodiments the biopolymer is a polysaccharide and the molar ratio of the catalyst to anhydrous glucose units (AGU) of the biopolymer particles is from about 1 :10 to about 1 :4. The foregoing ratios are calculated based on the number of moles of the first compound and the anhydrous glucose units prior to the reaction of said first compound and anhydrous glucose units, i.e. before the reactants are consumed / transformed. As used herein, “anhydrous glucose unit” takes its normal meaning in the art and refers to a single sugar molecule (i.e. monomer) in the polysaccharide of the biopolymer particles.

[0132] The molar ratio of the first compound to biopolymer particles is not necessarily limited. In various embodiments, the biopolymer is a polysaccharide and the molar ratio of the first compound to anhydrous glucose units (AGU) of the biopolymer particles is from about 1 :1 to about 10:1. In various embodiments, the molar ratio of the first compound to anhydrous glucose units (AGU) of the biopolymer particles is from about 1 : 1 to about 9:1 , from about 1 : 1 to about 8: 1 , from about 1 : 1 to about 7: 1 , or from about 1 : 1 to about 6: 1 . The foregoing ratios are calculated based on the number of moles of the first compound and the anhydrous glucose units prior to the reaction of said first compound and anhydrous glucose units, i.e. before the reactants are consumed / transformed. As used herein, “anhydrous glucose unit” takes itsnormal meaning in the art and refers to a single sugar molecule (i.e. monomer) in the polysaccharide of the biopolymer particles.Reaction in solvent (Method A)

[0133] In various embodiments, the reaction mixture for the esterification reaction comprises an organic solvent. In some embodiments, the organic solvent may be the same organic solvent in which the biopolymer particles are prepared, e.g. in which the biopolymer particles are dispersed, or with which solvent exchange is conducted as explained hereinabove.

[0134] In preferred embodiments, wherein the solvent dispersion of biopolymer particles is aqueous, the method further comprises subjecting the biopolymer particles to a solvent exchange prior to the esterification reaction, and wherein the solvent exchange is with an organic solvent, the organic solvent for the esterification reaction is thus preferably the same organic solvent with which the solvent exchange is performed. The aqueous solvent of the dispersion of biopolymer particles being as defined above.

[0135] In various embodiments, the organic solvent for the esterification reaction is preferably anhydrous. As used herein, the term “anhydrous” means substantially free of water. For example, the anhydrous solvent may comprise less than 100 ppm water, less than 10 ppm water or less than 1 ppm water. Anhydrous solvents are commercially available and also readily obtained by methods commonly known in the art such via drying trains, solvent stills, and / or the use of desiccants and drying agents such as molecular sieves. Water content is also readily determined by methods commonly known in the art such as Karl Fischer titration.

[0136] The reaction mixture may comprise an organic solvent and a nucleophilic catalyst. Thus, in various embodiments the organic solvent may be selected from acetone, acetonitrile, benzene, 1 -butanol, 2-butanol, 2-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1 ,2-dichloroethane, diethylene glycol, diethyl ether, diethylene glycol dimethyl ether, 1 ,2-dimethoxy ethane (DME), dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), 1 ,4-dixoane, ethanol, ethyl acetate, ethylene glycol, glycerol, heptane, hexamethylphosphoramide (HMPA), hexamethylphosphorous triamide (HMPT), hexane, methanol, methyl t-butyl ether (MTBE), methylene chloride, N-methyl-2-pyrrolidinone (NMP), nitromethane, pentane, petroleum ether, 1 -propanol, 2-propanol, pyridine, tetrahydrofuran (THF), toluene triethyl amine, xylene, and mixtures thereof and the nucleophilic catalyst is an amine compound, an N-heterocycle or an inorganic hydroxide salt, preferably an amine compound or an alkaline hydroxide salt selected from lithium hydroxide, potassium hydroxide and sodium hydroxide. In various embodiments, the nucleophilic catalyst is an acyclic or cyclic aliphatic amine, an aromatic amine, a six-membered N-heterocycle or analkaline hydroxide salt such as lithium hydroxide, potassium hydroxide or sodium hydroxide. In various embodiments, the nucleophilic catalyst is a trialkylamine, an amino-substituted N- heteroarene, or an alkaline hydroxide salt such as lithium hydroxide, potassium hydroxide or sodium hydroxide. In various embodiments, the nucleophilic catalyst is a Ci-Ce or C1-C3 trialkylamine, an amino-substituted N-heteroarene, or an alkaline hydroxide salt such as lithium hydroxide, potassium hydroxide or sodium hydroxide. In various embodiments, the nucleophilic catalyst is a Ci-Ce or C1-C3 trialkylamine, an amino-substituted N-heteroarene, or an alkaline hydroxide salt such as lithium hydroxide, potassium hydroxide or sodium hydroxide. In various embodiments, the nucleophilic catalyst is a Ci-Ce or C1-C3 trialkylamine, a Ci-Ce or C1-C3 dialkylamino-substituted N-heteroarene, or an alkaline hydroxide salt such as lithium hydroxide, potassium hydroxide or sodium hydroxide. In any of the preceding embodiments, the amine compound may be optionally further substituted. In various embodiments, the nucleophilic catalyst may be triethylamine, 4-dimethylaminopyridine (DMAP) or an alkaline hydroxide salt such as lithium hydroxide, potassium hydroxide or sodium hydroxide.

[0137] In various embodiments, the esterification reaction may comprise a reaction temperature of at least about 15 °C, at least about 20 °C, at least about 25 °C, at least about 30 °C, at least about 35 °C, at least about 40 °C, at least about 45 °C, at least about 50 °C, at least about 55 °C, at least about 60 °C, at least about 65 °C, or at least about 70 °C. The maximum reaction temperature is not necessarily limited. In various embodiments, the esterification reaction may comprise a reaction temperature less than about the boiling point of the first compound. Measurement methods for boiling point are part of the skilled person’s common general knowledge. For example, in various embodiments the esterification reaction comprises a reaction temperature less than about 120 °C. In various embodiments, the esterification reaction comprises a reaction temperature of from about 15 °C, about 20 °C, about 25 °C, about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C, about 65 °C or about 70 °C to about 120 °C.

[0138] In various embodiments, it may be preferable to minimise or avoid heating of the reaction mixture e.g. to minimise energy consumption. Additionally, or alternatively, it may be desirable to use moderate reaction conditions, e.g. reaction temperature, to minimise crosslinking. In various embodiments, it is desirable to minimise cross-linking as this may reduce the available chemical functionality for e.g. enzyme immobilisation. Additionally or alternatively, cross-linking may adversely affect one or more mechanical properties of the functionalised biopolymer particles.

[0139] Thus, in various embodiments, the esterification reaction comprises a reaction temperature less than about 110 °C, less than about 100 °C, less than about 90 °C, less thanabout 80 °C, less than about 70 °C, less than about 60 °C, less than about 50 °C, less than about 40 °C, or less than about 30 °C. For example, in various embodiments the esterification reaction comprises a reaction temperature of from about 15 °C to about 110 °C, about 100 °C, about 90 °C, about 80 °C, about 70 °C, about 60 °C, about 50 °C, about 40 °C, or about 30 °C.

[0140] In various embodiments, the esterification reaction comprises a reaction temperature of from about 15 °C to about 100 °C, from about 20 °C to about 90 °C or from about 30 °C to about 80 °C.

[0141] In various embodiments, an indirect heating means is employed for the esterification reaction. An indirect heating means such as an oil bath can be beneficial vis-a-vis a direct heating means such as microwave irradiation because of the ability to achieve moderate reaction conditions including e.g. speed of heating. In various embodiments, the esterification reaction is carried out in the absence of microwave irradiation.

[0142] In any of the foregoing embodiments, the reaction mixture may be mixed, stirred or otherwise agitated by any known means in the art.

[0143] The reaction time is not necessarily limited. A person of skill in the art will be able to determine a suitable duration for the reaction, for example to ensure the reaction reaches completion or to achieve varying degrees of partial functionalisation. For example, reaction progress can be monitored by taking performing a time course wherein samples of the reaction are taken at regular intervals, quenched, and the degree of functionalisation may be measured by any of the methods described herein such as FTIR spectroscopy, conductometric titration, and / or zeta potential measurement.Solvent-free reaction (Method B)

[0144] In other embodiments of the present disclosure, the biopolymer particles are esterified without being present in a solvent; the solvent of the biopolymer particle dispersion is removed prior to the esterification reaction. For example, the biopolymer particle dispersion may be filtered prior to the esterification reaction. When the solvent of the dispersion is aqueous, the method may in various embodiments thus further comprise subjecting the biopolymer particles to a solvent exchange prior to the esterification reaction, and filtering the biopolymer particles prior to the esterification reaction so as to remove the solvent. Preferably, the solvent exchange is with an organic solvent. In the foregoing embodiments, the biopolymer particles are preferably in the form of a hydrogel. In any of the foregoing embodiments, the solvent exchange and / or filtration may be performed to reduce the amount of water from the aqueous solvent of the dispersion carried over into the reaction mixture for esterification, particularlywhen the reaction mixture comprises an organic solvent such as those described herein. Thus, the organic solvent with which the solvent exchange is performed may, in various embodiments, preferably be an organic solvent that is miscible with water. In the foregoing embodiments, the reaction mixture for esterification comprises the biopolymer particles and the first compound but no solvent is added to said reaction mixture. Such embodiments may be advantageous in that the method may be performed with reduced consumption of organic solvent.

[0145] Reduced consumption of organic solvent as noted in the preceding paragraph and herein may be advantageous in terms of improved cost efficiency and / or improved environmental impact. Further advantages may include improved process scalability and reduced waste management requirements. Carrying out esterification without the addition of organic solvent may also enable the minimisation or avoidance of organic solvents whose presence in the functionalised biopolymer particle product may be undesirable (e.g. due to safety and / or regulatory requirements) and / or incompatible with downstream applications.

[0146] As already described herein above, in other aspects and embodiments, the biopolymer particles are dried prior to the esterification reaction. In various embodiments, the drying may be performed after a solvent exchange step and / or a filtration step such as those described herein above. Thus, in various embodiments, the method of the first aspect further comprises subjecting the biopolymer particles to a solvent exchange prior to the esterification reaction, and filtering the biopolymer particles prior to the esterification reaction; preferably wherein the solvent exchange is with an organic solvent.

[0147] In various embodiments, the drying is carried out to remove substantially all solvent from the biopolymer particles. For example, the biopolymer particles are dried at room temperature and / or at a temperature of from about 25 °C to about 90 °C, from about 35 °C to about 90 °C, or from about 45 °C to about 90 °C.

[0148] In various embodiments, drying involves one or more drying steps. For example, drying may comprise drying at a first temperature (e.g. room temperature) and drying at a second temperature, wherein the second temperature is higher than the first temperature by at least about 5°C, preferably by at least about 10°C. In various embodiments, the biopolymer particles are dried at room temperature and dried at a temperature of from about 25 °C to about 90 °C, from about 35 °C to about 90 °C, or from about 45 °C to about 90 °C.

[0149] In various embodiments, drying may comprise drying at a first temperature for a first period of time and drying at a second temperature for a second period of time, wherein the second temperature is higher than the first temperature by at least about 5°C, preferably by atleast about 10°C. In various embodiments, the biopolymer particles are dried at room temperature for at least 2, 4, 6, 8, 10, 12, 14 or 16 hours. In various embodiments, the biopolymer particles are dried at a temperature of from about 25 °C to about 90 °C, from about 35 °C to about 90 °C, or from about 45 °C to about 90 °C for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22 hours. Thus, in further embodiments, the biopolymer particles are dried at room temperature for at least 2, 4, 6, 8, 10, 12, 14 or 16 hours and dried at a temperature of from about 25 °C to about 90 °C, from about 35 °C to about 90 °C, or from about 45 °C to about 90 °C for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22 hours.

[0150] The means by which drying is conducted are not limited and would be known to a person skilled in the art. In various embodiments, the drying at the second temperature (e.g. for a second period of time as described herein above) is carried out using oven drying.

[0151] In further advantageous embodiments, it has been unexpectedly found that esterification may be performed in the absence of a nucleophilic catalyst, including those described herein. This may be advantageous by reducing the number and quantity of reagents required for the process, simplifying the process and / or reducing cost, and / or avoiding the use of reagents with undesirable safety and / or environmental profiles. For instance, 4- dimethylaminopyridine (DMAP) has a relatively high toxicity and is readily absorbed through the skin as well as being corrosive. Hence, in various embodiments it may be beneficial to perform the esterification in the absence of a nucleophilic catalyst such as an amine catalyst, e.g. DMAP.

[0152] Thus, in various embodiments, the reaction mixture for esterification consists essentially of the biopolymer particles and the first compound. As used herein, “consists essentially of” or “consisting essentially of” means that that the reaction mixture includes the recited, mandatory components, along with other components which do not materially affect the essential characteristics of said reaction mixture, e.g. minor amounts of impurities. Notably, the expression “consists essentially of” or “consisting essentially of” excludes the addition of a nucleophilic catalyst and the addition of solvent to the reaction mixture.

[0153] In further embodiments, the reaction mixture for esterification consists of the biopolymer particles and the first compound. As used herein, the expression “consists of” or “consisting of” means that the reaction mixture only includes the recited, mandatory components.

[0154] A person of skill in the art will recognise that the biopolymer particles may comprise residual amounts of substances such as from the biopolymer particle preparation process (e.g. water, particularly wherein the biopolymer particles are in the form of a hydrogel) and / or stepsperformed prior to esterification such as solvent exchange and / or filtration. For instance, the skilled person will recognise that the biopolymer particles may in some embodiments comprise amounts of solvent and / or other substances adsorbed to said particles. Neither the term “consists essentially of” nor “consists of” as used herein exclude the presence of such substances, which are to be understood to comprise part of the biopolymer particles as such.

[0155] In the foregoing embodiments, the first compound is preferably present as a liquid phase in the reaction mixture. In such embodiments the biopolymer particles may preferably be dispersed in the liquid phase of the first compound. This may be advantageous for example in ensuring efficient and thorough contact between the biopolymer particles and the first compound during the esterification reaction. In various embodiments, the esterification reaction comprises a reaction temperature that is at least the melting point of the first compound. Measurement methods for melting point are known in the common general knowledge. For example, the first compound may be liquid at ambient temperature and pressure, or the reaction mixture may be heated so as to comprise a reaction temperature that is at least the melting point of the first compound.

[0156] In various embodiments, the esterification reaction may comprise a reaction temperature of at least about 15 °C, at least about 20 °C, at least about 25 °C, at least about 30 °C, at least about 35 °C, at least about 40 °C, at least about 45 °C, at least about 50 °C, at least about 55 °C, at least about 60 °C, at least about 65 °C, or at least about 70 °C. The maximum reaction temperature is not necessarily limited. In various embodiments, the esterification reaction may comprise a reaction temperature less than about the boiling point of the first compound. For example, in various embodiments the esterification reaction comprises a reaction temperature less than about 120 °C. In various embodiments, the esterification reaction comprises a reaction temperature of from about 15 °C, about 20 °C, about 25 °C, about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C, about 65 °C or about 70 °C to about 120 °C. In any of the foregoing embodiments, the first compound is preferably present as a liquid phase.

[0157] In various embodiments, it may be preferable to minimise or avoid heating of the reaction mixture e.g. to minimise energy consumption. Additionally, or alternatively, it may be desirable to use moderate reaction conditions, e.g. reaction temperature, to minimise crosslinking. Thus, in various embodiments, the esterification reaction comprises a reaction temperature less than about 110 °C, less than about 100 °C, less than about 90 °C, less than about 80 °C, less than about 70 °C, less than about 60 °C, less than about 50 °C, less than about 40 °C, or less than about 30 °C. For example, in various embodiments the esterification reaction comprises a reaction temperature of from about 15 °C to about 110 °C, about 100 °C,about 90 °C, about 80 °C, about 70 °C, about 60 °C, about 50 °C, about 40 °C, or about 30 °C. In any of the foregoing embodiments, the first compound is preferably present as a liquid phase.

[0158] In various embodiments, the esterification reaction comprises a reaction temperature of from about 15 °C to about 100 °C, from about 20 °C to about 90 °C or from about 30 °C to about 80 °C.

[0159] In various embodiments, an indirect heating means is employed for the esterification reaction. An indirect heating means such as an oil bath can be beneficial vis-a-vis a direct heating means such as microwave irradiation because of the ability to achieve moderate reaction conditions including e.g. speed of heating. In various embodiments, the esterification reaction is carried out in the absence of microwave irradiation.

[0160] In any of the foregoing embodiments, the reaction mixture may be mixed, stirred or otherwise agitated by any known means in the art.

[0161] The reaction time is not necessarily limited. A person of skill in the art will be able to determine a suitable duration for the reaction, for example to ensure the reaction reaches completion or to achieve varying degrees of partial functionalisation. For example, reaction progress can be monitored by taking performing a time course wherein samples of the reaction are taken at regular intervals, quenched, and the degree of functionalisation may be measured by any of the methods described herein such as FTIR spectroscopy, conductometric titration, and / or zeta potential measurement.

[0162] In a sixth aspect, the present disclosure provides a method for preparing functionalised biopolymer particles from a solvent dispersion of biopolymer particles, wherein the solvent of the dispersion is aqueous, said method comprising: optionally subjecting the solvent dispersion of biopolymer particles to a solvent exchange; optionally filtering the biopolymer particles; drying the biopolymer particles to remove the solvent; mixing the dried biopolymer particles with a first compound, wherein the first compound comprises at least one carboxyl group or is an acid anhydride, and reacting the biopolymer particles with the first compound via an esterification reaction to form the functionalised biopolymer particles.It will be understood that the method of the sixth aspect may be combined with any of the embodiments disclosed herein, including those described in the foregoing paragraphs.

[0163] For example, in various embodiments the solvent exchange is with an organic solvent.

[0164] In various embodiments of the sixth aspect, the drying is carried out to remove substantially all solvent from the biopolymer particles. For example, the biopolymer particles are dried at room temperature and / or at a temperature of from about 25 °C to about 90 °C, from about 35 °C to about 90 °C, or from about 45 °C to about 90 °C.

[0165] In various embodiments of the sixth aspect, drying involves one or more drying steps. For example, drying may comprise drying at a first temperature (e.g. room temperature) and drying at a second temperature, wherein the second temperature is higher than the first temperature by at least about 5°C, preferably by at least about 10°C. In various embodiments, the biopolymer particles are dried at room temperature and dried at a temperature of from about 25 °C to about 90 °C, from about 35 °C to about 90 °C, or from about 45 °C to about 90 °C.

[0166] In various embodiments of the sixth aspect, drying may comprise drying at a first temperature for a first period of time and drying at a second temperature for a second period of time, wherein the second temperature is higher than the first temperature by at least about 5°C, preferably by at least about 10°C. In various embodiments, the biopolymer particles are dried at room temperature for at least 2, 4, 6, 8, 10, 12, 14 or 16 hours. In various embodiments, the biopolymer particles are dried at a temperature of from about 25 °C to about 90 °C, from about 35 °C to about 90 °C, or from about 45 °C to about 90 °C for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22 hours. Thus, in further embodiments, the biopolymer particles are dried at room temperature for at least 2, 4, 6, 8, 10, 12, 14 or 16 hours and dried at a temperature of from about 25 °C to about 90 °C, from about 35 °C to about 90 °C, or from about 45 °C to about 90 °C for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22 hours.

[0167] In various embodiments of the sixth aspect, the drying at the second temperature (e.g. for a second period of time as described herein above) is carried out using oven drying.

[0168] In various embodiments of the sixth aspect, the solvent exchange may be omitted. For example, in various embodiments the biopolymer particles may be dried to remove the aqueous solvent. Suitable drying methods for removing the aqueous solvent are commonly known in the art and non-limiting examples include spray drying, microwave drying, fluidised- bed drying and freeze drying. In further embodiments, the biopolymer particles may be filtered prior to drying. The skilled person is able to select suitable parameters for such methods, e.g. drying temperature, as part of their common general knowledge. In various embodiments wherein the biopolymer particles are dried to remove the aqueous solvent, the beads may be dried at a temperature of at least about 100 °C. For example, the biopolymer particles may invarious embodiments by dried by spray drying, wherein the spray drying comprises a drying temperature of at least about 100 °C.

[0169] In various embodiments of the sixth aspect, the esterification reaction is carried out in the absence of a nucleophilic catalyst such as an amine catalyst, e.g. DMAP.

[0170] Thus, in various embodiments of the sixth aspect, the reaction mixture for esterification consists essentially of the biopolymer particles and the first compound as already defined herein above.

[0171] In further embodiments of the sixth aspect, the reaction mixture for esterification consists of the biopolymer particles and the first compound as already defined herein above.

[0172] In the foregoing embodiments of the sixth aspect, the first compound is preferably present as a liquid phase in the reaction mixture. In such embodiments the biopolymer particles may preferably be dispersed in the liquid phase of the first compound. This may be advantageous for example in ensuring efficient and thorough contact between the biopolymer particles and the first compound during the esterification reaction. In various embodiments, the esterification reaction comprises a reaction temperature that is at least the melting point of the first compound. Measurement methods for melting point are known in the common general knowledge. For example, the first compound may be liquid at ambient temperature and pressure, or the reaction mixture may be heated so as to comprise a reaction temperature that is at least the melting point of the first compound.

[0173] In various embodiments of the sixth aspect, the esterification reaction may comprise a reaction temperature of at least about 15 °C, at least about 20 °C, at least about 25 °C, at least about 30 °C, at least about 35 °C, at least about 40 °C, at least about 45 °C, at least about 50 °C, at least about 55 °C, at least about 60 °C, at least about 65 °C, or at least about 70 °C. The maximum reaction temperature is not necessarily limited. In various embodiments, the esterification reaction may comprise a reaction temperature less than about the boiling point of the first compound. For example, in various embodiments the esterification reaction comprises a reaction temperature less than about 120 °C. In various embodiments, the esterification reaction comprises a reaction temperature of from about 15 °C, about 20 °C, about 25 °C, about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C, about 65 °C or about 70 °C to about 120 °C. In any of the foregoing embodiments, the first compound is preferably present as a liquid phase.

[0174] In various embodiments of the sixth aspect, it may be preferable to minimise or avoid heating of the reaction mixture e.g. to minimise energy consumption. Additionally, oralternatively, it may be desirable to use moderate reaction conditions, e.g. reaction temperature, to minimise cross-linking. Thus, in various embodiments, the esterification reaction comprises a reaction temperature less than about 110 °C, less than about 100 °C, less than about 90 °C, less than about 80 °C, less than about 70 °C, less than about 60 °C, less than about 50 °C, less than about 40 °C, or less than about 30 °C. For example, in various embodiments the esterification reaction comprises a reaction temperature of from about 15 °C to about 110 °C, about 100 °C, about 90 °C, about 80 °C, about 70 °C, about 60 °C, about 50 °C, about 40 °C, or about 30 °C. In any of the foregoing embodiments, the first compound is preferably present as a liquid phase.

[0175] In various embodiments of the sixth aspect, the esterification reaction comprises a reaction temperature of from about 15 °C to about 100 °C, from about 20 °C to about 90 °C or from about 30 °C to about 80 °C.

[0176] In various embodiments of the sixth aspect, an indirect heating means is employed for the esterification reaction. For example, in various embodiments, the esterification reaction is carried out in the absence of microwave irradiation.

[0177] In any of the foregoing embodiments, the reaction mixture may be mixed, stirred or otherwise agitated by any known means in the art.

[0178] In various embodiments of the sixth aspect, the biopolymer may be according to any of the embodiments described herein. For example, in various embodiments of the sixth aspect the biopolymer is a polysaccharide, preferably wherein the biopolymer is selected from cellulose, starch, agarose, dextran, chitosan, pectin and mixtures thereof. In further embodiments, the biopolymer is cellulose, preferably regenerated cellulose.

[0179] In various embodiments of the sixth aspect, the biopolymer particles may be in the form of beads as already defined herein.

[0180] In various embodiments of the sixth aspect, the first compound may be according to any of the embodiments described herein. For example, in various embodiments the first compound is selected from a monocarboxylic acid, a dicarboxylic acid, and an acid anhydride, preferably a cyclic anhydride. In further embodiments, the first compound is a C3-C26 compound or is unsaturated, preferably wherein the first compound is an unsaturated C3-C26 compound.Further processing and storage of functionalised beads

[0181] In various embodiments of the present disclosure, the methods disclosed herein further comprise separating the functionalised beads by filtration and optionally washing the filtrate with a solvent. The filtration process is not limited. Techniques suitable for the separation will be known to a person skilled in the art and include but are not limited to those described herein. For example, the functionalised biopolymer particles may be separated from the reactants by vacuum filtration or simply by sieving. The filtration process 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 reactants and thereby collect the biopolymer particles.

[0182] In various embodiments, the functionalised biopolymer particles may be allowed to settle in a vessel and the other components of the reaction mixture removed or decanted to leave the functionalised biopolymers. Alternatively, the biopolymer particles may be separated by a centrifugal separator or a disk stack separator.

[0183] In various embodiments, the functionalised biopolymer particles may be washed one or more times with a solvent. In various embodiments, the solvent is the same solvent as present for the esterification reaction, e.g. an organic solvent such as those described herein. In such embodiments, particularly wherein the functionalised biopolymer particles are washed with an organic solvent, the functionalised biopolymer particles are preferably then solvent exchanged into an aqueous solvent for further downstream steps.

[0184] In other embodiments wherein a solvent is not added to the reaction mixture for esterification, e.g. wherein the reaction mixture consists essentially of the biopolymer particles and the first compound, preferably wherein the reaction mixture consists of the biopolymer particles and the first compound, the washing solvent is preferably aqueous. In such embodiments, the solvent is preferably at a temperature that is at least the melting point of the first compound.

[0185] One or more cycles of separation and washing steps may be performed, preferably until the remaining amounts of reactants and / or any side-products from the reaction are negligible. For example, in various embodiments at least one, at least two or at least three washing steps may be performed.

[0186] In the foregoing embodiments, the first wash, e.g. with an aqueous solvent, may be collected. The collected washing solvent may be cooled, e.g. to a temperature at or below ambient temperature such as less than 20 °C. In such embodiments the temperature of thecollected washing solvent may be maintained until any unconsumed first compound present in the collected washing solvent crystallises. Advantageously, the crystallised first compound may then be recovered, optionally regenerated (e.g. by converting a dicarboxylic acid to a cyclic anhydride by means commonly known in the art of organic chemistry), and reused. In such embodiments, wastage of the first compound may be minimised, further improving the cost-effectiveness and sustainability of the method of the present disclosure.

[0187] In various embodiments, the functionalised beads are stored in an aqueous solvent, preferably wherein said aqueous solvent is substantially free of organic solvents. The term “substantially free” and “aqueous solvent” being as defined above. For example, the functionalised beads may be stored in water, preferably deionised water. In such embodiments, the functionalised beads may preferably be stored at temperatures lower than room temperature, for example less than about 20 °C. For example, functionalised beads may be stored in deionised water at less than about 10 °C. In various embodiments, the functionalised beads are stored in deionised water at about 4 °C. In various embodiments, the functionalised beads are stored in the manner according to any of the foregoing embodiments prior to use as described herein. In such embodiments, this may be advantageous to remove the need for additional drying and swelling steps as discussed below. In various embodiments where the avoidance of microbial contamination is required, the functionalised beads may be sterilised prior to storage as described herein. Alternatively or additionally, the functionalised beads may be sterilised prior to their subsequent use as described herein. The present disclosure is not limited in terms of suitable sterilisation methods and the skilled person will be able to select suitable sterilisation methods. For example, the functionalised beads may be sterilised by irradiation (e.g. UV- irradiation or gamma-ray irradiation), heat treatment (autoclavation), or chemical disinfection (e.g. 70% v / v ethanol in water).

[0188] In other embodiments, the functionalised beads may be dried (for example in a vacuum or microwave oven, by air drying, in a rotovaporator or by freeze-drying) and stored in a dry state. Such dried beads may be rehydrated by suspension in an aqueous solvent prior to use in a desired application as described herein. In various embodiments, the aqueous solvent is substantially free of organic solvents. The term “substantially free” and “aqueous solvent” being as defined above. As already discussed above, avoiding organic solvents may be advantageous in that the functionalised beads produced by the methods disclosed herein may be immediately suitable for use in downstream applications without further costly purification steps.Enzyme immobilisation

[0189] The functionalised biopolymer particles of the present disclosure have been found to be particularly suitable as substrates for the immobilisation of enzymes. Thus, in a second aspect the present disclosure provides a method for immobilising enzymes on functionalised biopolymer particles, wherein the method comprises preparing functionalised particles by the method of the first aspect described herein and contacting the functionalised particles with an enzyme. The functionalised particles may also be prepared by the method of the sixth aspect as described herein.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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. In particular, 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. Similarly, functionalisation via the esterification reaction described herein can minimise the use of hazardous or environmentally harmful substances such as organic solvents and toxic reagents, and can allow the recovery and re-use of unconsumed reactants.As already described herein, the methods of the present disclosure are particularly advantageous over prior art methods in that functionalisation requires a single reaction step, can use bio-derived starting materials, and can avoid or minimise the use of organic solvents and / or reagents with undesirable safety and / or environmental profiles.

[0194] 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 oxidoreductases, aminotransferases and lipases. For example, in various embodiments the enzyme may be a glucose oxidase, a horseradish peroxidase, a glucose isomerase, a transaminase or a lipase. Such enzymes are known and / or commercially available and the selection and / or production of said enzymes is specifically within the common general knowledge of a person skilled in the art. For example, in various embodiments the enzyme may be purified from an organism, e.g. a microorganism, that endogenously produces said enzyme. In various embodiments, the lipase is recombinantly produced in a heterologous microorganism.

[0195] In various embodiments of the present disclosure, the functionalised 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.

[0196] 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.

[0197] In various embodiments of the present disclosure, the functionalised biopolymer particles are contacted with an enzyme which is an oxidoreductase or an intramolecular oxidoreductase. Oxidoreductases are enzymes that catalyse the transfer of electrons from one molecule, the reductant or electron donor, to another molecule, the oxidant or electron acceptor. Intramolecular oxidoreductases are enzymes that catalyse the structural rearrangement of isomers by oxidising one part of a molecule while reducing another part of the same molecule. Many are known and / or commercially available and the selection and / or production of such enzymes by conventional means is specifically within the common general knowledge of a person skilled in the art.

[0198] Oxidoreductases are classified as EC 1 in the EC number classification of enzymes. They can be further classified into 21 subclasses. In various embodiments, the oxidoreductase enzyme is selected from the EC 1.1 or EC 1.11 subclass, namely those acting on the CH-OH group of donors or those acting on peroxide as an acceptor. In preferred embodiments, the oxidoreductase enzyme is selected from the EC 1.1.3 subclass, namely those acting on the CH-OH group of donors with oxygen as the acceptor. Suitable enzymes in the EC 1.1.3 subclass are known in the art, examples include glucose oxidase. Glucose oxidase is widely used for the determination of free glucose in body fluids, in vegetal raw material and in the food industry. It is commercially available from e.g. Sigma Aldrich. In other preferred embodiments, the oxidoreductase enzyme is selected from the EC 1.11.1.7 subclass, namely peroxidase, this enzyme is a protein target, examples include horseradish peroxidase which is used extensively in biochemistry applications.

[0199] Intramolecular oxidoreductases are classified as EC 5.3 in the EC number classification of enzymes. They can be further classified into 5 subclasses. In various embodiments, the intramolecular oxidoreductase enzyme is selected from the EC 5.3.1 subclass, namely those that interconvert aldoses and ketoses. Suitable enzymes in the EC 5.3.1 subclass are known in the art, examples include glucose isomerase, otherwise known as xylose isomerase. Glucose isomerase is an enzyme that catalyses the interconversion of D-xylose and D- xylulose. It is widely used in the conversion of glucose to fructose to produce high fructose corn syrup, and identified as being possible useful in the production of biofuel. It is commercially available from e.g. Sigma Aldrich.

[0200] Transaminases are otherwise known as aminotransferases and are enzymes that catalyse a transamination reaction between an amino acid and an alpha-keto acid. They are important in the synthesis of amino acids and measuring the concentrations of various transaminases in the blood is important in the diagnosis and tracking of many diseases. Transaminases are classified as EC 2.6.1 in the EC number classification of enzymes.

[0201] As discussed above, it has been found that the functionalised biopolymer particles of the present disclosure may be used for the immobilisation of enzymes. Thus, in various embodiments, upon contacting the functionalised biopolymer particles with an enzyme, the enzyme is immobilised on said biopolymer particles.

[0202] Without wishing to be bound by theory, the immobilisation may be mediated by covalent and / or 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-effectssuch as pi-pi, cation-pi or anion-pi interactions, and combinations thereof. In various embodiments, the enzyme is covalently immobilised on the particles. In particular, in various embodiments wherein the first compound is unsaturated, e.g. wherein the first compound comprises an alkene bond, the enzyme may be covalently immobilised through reaction with an alkene bond on the functionalised biopolymer particles. For example, the alkene bond functionality of the biopolymer particles derived from the first compound as described herein may in various embodiments (and without wishing to be bound by theory) be able to act as a Michael acceptor for nucleophilic side-chain groups comprised in the enzyme being immobilised, thereby leading to covalent bonding of the enzyme to the biopolymer particles. In such embodiments, the enzyme may preferably be immobilised in the presence of a base. Covalent immobilisation may be advantageous in that the enzyme functionalised biopolymer particles may be more durable when compared to non-covalent immobilisation alone, e.g. the enzyme functionalised biopolymer particles may show higher retention of bound protein such as after washing and recycling steps or other stringent conditions.

[0203] The 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 (see e.g. Example 5 and Figure 4; Example 6 and Figure 5). Moreover, the enzyme functionalised biopolymer particles of the present disclosure exhibit good retention of adsorbed enzyme (see e.g. Example 5 and Figure 4). Further advantages of the functionalised biopolymer particles of the present disclosure may include good storage stability, for example 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 about 4 °C.

[0204] 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, the method comprising the step of forming a reaction mixture comprising functionalised biopolymer particles as described herein in respect of the second aspect and at least one substrate. The nature of the reaction is not limited, and the skilled person will be able to select enzymes and substrates capable of performing the desired chemical transformation. The reaction may be performed in aqueous solvent or may be performed in one or more organic solvents. The selection of suitable solventsystems and other reaction conditions for performing a biocatalytic reaction is also in the common general knowledge of a person skilled in the art.Preparation of biopolymer particles

[0205] 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.

[0206] 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.

[0207] 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.

[0208] The latter exemplary method of preparing biopolymer particles comprises a membrane emulsification step and a phase inversion step.

[0209] 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.

[0210] 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 theirinhomogeneous mixture is termed an emulsion, i.e. droplets of the dispersed phase surrounded by the continuous phase.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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],

[0216] Turning 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.

[0217] 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 biopolymer particle sphericity.

[0218] 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.

[0219] 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).

[0220] 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, tetra hydrofuran, 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'- tetramethyl urea, 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.

[0221] 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.

[0222] 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. Thus, for polysaccharide such as cellulose, starch, chitin, glycogen, and / or chitosan, the solvent for the dispersed phase may 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 co-solvents. Each of these disclosures is incorporated herein by reference.

[0223] 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.

[0224] 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 andcarboxylates 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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°Cto 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

[0229] 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.

[0230] 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.

[0231] 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%.

[0232] 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.

[0233] 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 atemperature between about 20 and about 25°C. Alternatively, the anti-solvent may be cooled to a temperature below ambient temperature, namely below about 20°C. For example, the antisolvent 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.(i) Extrusion

[0234] 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.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] 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 thanabout 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 greaterthan about O.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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] The maximum dropping height will typically 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 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.

[0245] 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.

[0246] 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 extrudedphase 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.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] In various embodiments, the biopolymer particles may be washed one or more times, for example with an aqueous solvent including water. In various embodiments, the solvent in which the biopolymer particles are immersed may be exchanged for an alternative solvent.(ii) Membrane emulsification

[0251] 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 structuresuitable 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.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] The surfactant is as defined above.

[0263] In various embodiments, the emulsion is cooled to a temperature Ti , Ti being greater than the pour point of the continuous phase (Tcont), 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 > TCOnt. 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.

[0264] 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.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] 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 thatthe 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).

[0269] 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.

[0270] 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.

[0271] 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 .

[0272] The temperature of the anti-solvent during phase inversion is discussed above.

[0273] 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.

[0274] 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.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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).

[0279] 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 heatexchanger 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.

[0280] 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.

[0281] 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.Numbered clauses

[0282] Further exemplary embodiments are now described in the following numbered clauses.1 . A method for preparing functionalised biopolymer particles, said method comprising: mixing a solvent dispersion of biopolymer particles with a first compound, wherein the first compound comprises at least one carboxyl group or is an acid anhydride, and reacting the biopolymer particles with the first compound via an esterification reaction to form the functionalised biopolymer particles.2. The method according to clause 1 , wherein the biopolymer is a polysaccharide, preferably wherein the biopolymer is selected from cellulose, starch, agarose, dextran, chitosan, pectin and mixtures thereof.3. The method according to clause 1 or clause 2, wherein the biopolymer is cellulose, preferably wherein the cellulose is regenerated cellulose.4. The method according to any preceding clause, wherein the solvent of the dispersion is aqueous and the biopolymer particles are in the form of a hydrogel, and wherein the method further comprises subjecting the biopolymer particles to a solvent exchange prior to the esterification reaction, preferably wherein the solvent exchange is with an organic solvent.5. The method according to any preceding clause, wherein the biopolymer particles are in the form of beads.6. The method according to any preceding clause, wherein the first compound is selected from a monocarboxylic acid, a dicarboxylic acid, and an acid anhydride, preferably wherein the acid anhydride is a cyclic anhydride.7. The method according to clause 6, wherein the first compound is a C3-C26 compound, or wherein the first compound is unsaturated, preferably wherein the first compound is an unsaturated C3-C26 compound.8. The method according to any preceding clause, wherein the first compound is selected from itaconic acid, itaconic anhydride and mixtures thereof.9. The method according to any preceding clause, wherein the reaction mixture comprises an organic solvent.10. The method according to clause 9, wherein the reaction mixture further comprises a nucleophilic catalyst; preferably wherein the nucleophilic catalyst is an amine compound, an N-heterocycle, an inorganic hydroxide salt, or a combination thereof.11. The method according to any preceding clause, wherein the esterification reaction comprises a reaction temperature of from about 15 °C to about 120 °C.12. The method according to any one of clauses 1 to 8, wherein the solvent of the dispersion is aqueous and the biopolymer particles are in the form of a hydrogel, wherein the method further comprises subjecting the biopolymer particles to a solvent exchange prior to the esterification reaction, and filtering the biopolymer particles prior to the esterification reaction; preferably wherein the solvent exchange is with an organic solvent.13. The method according to any one of clauses 1 to 8 and clause 12, wherein the esterification reaction occurs in the absence of a nucleophilic catalyst.14. The method according to any one of clauses 1 to 8 and clause 12 or 13, wherein the reaction mixture consists essentially of the biopolymer particles and the first compound, preferably wherein the reaction mixture consists of the biopolymer particles and the first compound.15. The method according to any one of clauses 1 to 8 and clauses 12 to 14, wherein the first compound is present as a liquid phase.16. The method according to any one of clauses 1 to 8 and clauses 12 to 15, wherein the esterification reaction is carried out at a reaction temperature that is at least the melting point of the first compound.The method according to any preceding clause, wherein the biopolymer is a polysaccharide and the molar ratio of the first compound to anhydrous glucose units of the biopolymer particles is from about 1:1 to about 10:1. The method according to any preceding clause, wherein 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; optionally wherein 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. The method according to any one of clauses 1 to 17, wherein the biopolymer particles are prepared by: 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. A method for immobilising enzymes on functionalised biopolymer particles, wherein the method comprises preparing functionalised particles by the method of any one of clauses 1 to 19, and contacting the functionalised particles with an enzyme, preferably wherein the enzyme is a lipase, an oxidoreductase, or an aminotransferase. The method according to clause 20, wherein the enzyme is covalently immobilised on the particles. Functionalised biopolymer particles prepared by the method according to any preceding clause, preferably wherein the particles are approximately spherical and / or have a diameter from about 1 pm to about 3 mm. Use of the functionalised biopolymer particles according to clause 22 for immobilising an enzyme, preferably wherein the enzyme is a lipase, an oxidoreductase, or an aminotransferase. A method for performing an enzyme-catalysed reaction, the method comprising forming a reaction mixture comprising the enzyme immobilised functionalisedbiopolymer particles prepared by the method according to clause 20 or clause 21 , and at least one substrate.25. Use of enzyme immobilised functionalised biopolymer particles prepared by the method according to clause 20 or clause 21 for catalysing a reaction.

[0283] 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.ExamplesMaterials and methods

[0284] Itaconic anhydride (ITA, 98%) and 4-(Dimethylamino)pyridine (DMAP, 99%) were from Sigma-Aldrich (Merck KGaA). Isopropanol (IPA), tetra hydrofuran (THF) and ethyl acetate (EtOAC) were from VWR.Preparation of cellulose solutions

[0285] Microcrystalline cellulose (MCC, from Sigma-Aldrich®) and EmimOAc were dried in a vacuum oven at a temperature below 100°C until all water traces were removed. Cellulose solutions were prepared at a concentration of 4-10 wt% MCC in the EmimOAc with 4-10 wt% deionized water content. The ingredients were added under stirring. The mixture was agitated, then dried in an oven at a temperature below 100°C for 48 h, followed by agitation for further 24 h.Preparation of cellulose beads by extrusion

[0286] 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

[0287] A dispersed phase comprising 6 wt% microcrystalline cellulose and 8 wt% water in 1- ethyl-3-methylimidazolium acetate was prepared according to routine methods known in the art. An oily continuous phase was also prepared according to routine methods known in the art.

[0288] The dispersed phase and continuous phase were fed into a membrane emulsification unit and an emulsion thereby formed. The emulsion was then transferred into a phase inversion unit with an aqueous anti-solvent to form cellulose beads.Example 1 : Functionalisation of cellulose beads with itaconic anhydride in organic solvent (Method A)

[0289] Cellulose beads with average diameter 75-100 pm were prepared according to the methods described herein above and provided dispersed in deionised water. The beads were first solvent exchanged to THF. In this step, a predetermined amount of beads was filtered using vacuum filtration to remove excess water. These filtered beads were then weighed into an oven-dried round bottom flask with a magnetic stirrer, followed by addition of THF. The contents were stirred at room temperature. The beads were then vacuum filtered and redispersed into THF. The above process was repeated 3 times.

[0290] To the beads dispersed in THF, ITA (1 g, 3.0 equiv. with respect to anhydroglucose units, AGU, in dry cellulose) and DMAP (0.2 g, 0.25 equiv. to AGU) were added with constant stirring. The flask was then transferred to an oil bath maintained at 40-60 °C and allowed to react for 16-20 hours. The reaction was allowed to cool to room temperature after removing the flask from the oil bath. The functionalized cellulose beads were then vacuum filtered and washed with an excess of THF. This was followed by solvent exchanging the cellulose beads to DI water using the same procedure as described for THF solvent exchange. The final functionalized cellulose beads were dispersed in DI water for further use.Example 2: Functionalisation of cellulose beads with itaconic anhydride in the absence of solvent (Method B1)

[0291] Cellulose beads with average diameter 75-100 pm were prepared according to the methods described herein above and provided dispersed in deionised water. The beads were then vacuum filtered to remove excess water. From the filtrate, beads were weighed into an oven-dried round bottom flask with a magnetic stirrer. To remove water adsorbed to the beads, a solvent exchange step into IPA was performed in a similar manner to the solvent exchange described in Example 1 . The beads dispersed in IPA were then vacuum filtered and set aside.

[0292] Separately, ITA (6 g, 5.5 equiv. to AGU) was weighed into an oven-dried round bottom flask with a magnetic stirrer. The flask was heated to 50-70 °C using an oil bath to allow ITA to melt. Once ITA was completely melted, the beads prepared as described in the preceding paragraph were gradually added. After addition of the beads was complete, DMAP (0.25 equiv. to AGU) was added. The reaction mixture was allowed to react for 8-12 hours at 60-80 °C. Theflask was then removed from the oil bath. The functionalized beads were subsequently washed with hot water (60-80 °C) to remove unreacted ITA. The washed beads were then dispersed in DI water for further use.

[0293] Alternatively, the foregoing procedure was carried out but with the omission of DMAP.Example 3: Functionalisation of cellulose beads with itaconic anhydride in the absence of solvent (Method B2)

[0294] Cellulose beads with average diameter 75-100 pm were prepared according to the methods described herein above and provided dispersed in deionised water. The beads were then vacuum filtered to remove excess water. From the filtrate, beads were weighed into an oven-dried round bottom flask with a magnetic stirrer. To remove water adsorbed to the beads, a solvent exchange step into I PA was performed in a similar manner to the solvent exchange described in Example 1. The beads dispersed in I PA were then vacuum filtered and solvent gradually evaporated at ambient temperature for 18 hours. The beads were then transferred to an oven at 65 °C for 24 hours to ensure complete removal of solvent.

[0295] Separately, ITA (29 g, 3.5 equiv. to AGU) was weighed into an oven-dried two-neck round bottom flask fitted with an overhead stirring system. The flask was heated to 50-70 °C using an oil bath to allow ITA to melt. Once ITA was completely melted, the beads prepared as described in the preceding paragraph were gradually added. The reaction mixture was allowed to react for 5-10 hours at 60-80 °C with gentle stirring. The flask was then removed from the oil bath. The functionalized beads were subsequently washed with hot water (60- 80 °C) to remove unreacted ITA. The washed beads were then dispersed in DI water for further use.Example 4: Characterisation of functionalised cellulose beads by FTIR spectroscopy

[0296] Functionalised cellulose beads prepared according to each of Examples 1 ,2 and 3 were characterised by attenuated total reflectance Fourier transform infrared spectroscopy (ATR- FTIR). Representative spectra compared to unmodified cellulose beads are shown in Figure 3A for functionalised beads according to Example 1 , in Figure 3B for functionalised beads according to Example 2, and in Figure 3C for functionalised beads according to Example 3. Figures 3A, 3B and 3C show successful esterification of the cellulose beads in each case as determined by the presence of a peak at around 1715 cm-1that could be assigned to the carbonyl stretch (C=O) of the itaconate moieties resulting from the esterification of the cellulose beads. In contrast, the unmodified cellulose beads did not exhibit a peak at around 1715 cm-1. “NB-ITA ester 4” was functionalised with ITA in the presence of DMAP as a nucleophiliccatalyst. “NB-ITA ester 6” was functionalised with ITA in the absence of DMAP, with a reaction time of 5 h at 70 °C. “NB-ITA ester 7” was functionalised with ITA in the absence of DMAP, with a reaction time of 5 h at 80 °C.Example 5: Enzyme functionalisation of functionalised cellulose beads

[0297] Equal volumes of functionalised cellulose beads prepared according to Example 2, which had been stored in deionised water and not allowed to dry at any point, were immersed into a lipase solution having an enzyme concentration of 3-12 mg / mL. “NB-ITA ester 4” was functionalised with ITA in the presence of DMAP as a nucleophilic catalyst. “NB-ITA ester 6” was functionalised with ITA in the absence of DMAP. The resulting mixture was incubated at room temperature for 24 hours. The lipase used was Thermomyces lanuginosus lipase (TL Lipolase), which was obtained from ChiralVision B.V., Netherlands.

[0298] It was found that functionalisation with itaconic anhydride 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 functionalised cellulose beads. Thus, a lower amount of free enzyme in solution after incubation indicates a higher degree of adsorption.

[0299] As can be seen in Figure 4 (bars marked ‘uptake’), cellulose beads functionalised with ITA exhibited significantly greater adsorption of enzyme compared to unmodified cellulose beads. Further, cellulose beads functionalised with ITA exhibited significantly improved enzyme retention after washing with phosphate-buffered saline (PBS - see Figure 4 - bars marked ‘retention’). Retention was determined in a similar manner after washing with phosphate-buffered saline (PBS). A lower amount of free enzyme in the washing solution indicates a higher degree of enzyme retention on the beads.Example 6: Measurement of enzyme activity

[0300] 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: g p H+p-Nitrophenyl butyrate Water Butyric acid p-nitrophenol Hydrogen

[0301] About 0.6 mg of enzyme functionalised beads as prepared in Example 5 were suspended in 2 mL of Phosphate Buffered Saline (PBS, pH ~7.4) containing 1 mM of p- nitrophenyl butyrate. “NB-ITA ester 4” was functionalised with ITA in the presence of DMAP as a nucleophilic catalyst. 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 (II) 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 > [ A400nm / min Beads) - A400nm / min Blank)] g(Beads) £ x I x g(Beads) x 1000 wherein:A / oonm = absorbance (final) - absorbance (initial);E = 0.01725 pM-1cm’1;I = 1 cm; g(Beads) = dry weight of beads in grams.

[0302] As can be seen from Figure 5, the enzyme immobilised functionalised beads exhibited a significantly higher activity than for enzyme immobilised on unmodified beads.

[0303] 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.

Claims

CLAIMS1. A method for preparing functionalised biopolymer particles, said method comprising: mixing a solvent dispersion of biopolymer particles with a first compound, wherein the first compound comprises at least one carboxyl group or is an acid anhydride, and reacting the biopolymer particles with the first compound via an esterification reaction to form the functionalised biopolymer particles; wherein the solvent of the dispersion is aqueous and wherein the biopolymer particles are in the form of a hydrogel.

2. The method according to claim 1, wherein the biopolymer is a polysaccharide, preferably wherein the biopolymer is selected from cellulose, starch, agarose, dextran, chitosan, pectin and mixtures thereof.

3. The method according to claim 1 or claim 2, wherein the biopolymer is cellulose, preferably wherein the cellulose is regenerated cellulose.

4. The method according to any preceding claim, wherein the method further comprises subjecting the biopolymer particles to a solvent exchange prior to the esterification reaction, preferably wherein the solvent exchange is with an organic solvent.

5. The method according to any preceding claim, wherein the biopolymer particles are in the form of beads.

6. The method according to any preceding claim, wherein the first compound is selected from a monocarboxylic acid, a dicarboxylic acid, and an acid anhydride, preferably wherein the acid anhydride is a cyclic anhydride.

7. The method according to claim 6, wherein the first compound is a C3-C26 compound, or wherein the first compound is unsaturated, preferably wherein the first compound is an unsaturated C3-C26 compound.

8. The method according to any preceding claim, wherein the reaction mixture comprises an organic solvent, optionally wherein the reaction mixture further comprises a nucleophilic catalyst; preferably wherein the nucleophilic catalyst is an amine compound, an N-heterocycle, an inorganic hydroxide salt, or a combination thereof.

9. The method according to any preceding claim, wherein the esterification reaction comprises a reaction temperature of from about 15 °C to about 120 °C.

10. The method according to any one of claims 1 to 7, wherein the method further comprises subjecting the biopolymer particles to a solvent exchange prior to the esterification reaction, and filtering the biopolymer particles prior to the esterification reaction; preferably wherein the solvent exchange is with an organic solvent.11 . The method according to any one of claims 1 to 7 or claim 10, wherein the method further comprises drying the filtered biopolymer particles prior to the esterification reaction.

12. The method according to any one of claims 1 to 7 and claim 10 or 11 , wherein the esterification reaction occurs in the absence of a nucleophilic catalyst.

13. The method according to any one of claims 1 to 8 and claims 10 to 12, wherein the reaction mixture consists essentially of the biopolymer particles and the first compound, preferably wherein the reaction mixture consists of the biopolymer particles and the first compound.

14. The method according to any one of claims 1 to 8 and claims 10 to 13, wherein the first compound is present as a liquid phase.

15. The method according to any one of claims 1 to 8 and claims 10 to 14, wherein the esterification reaction is carried out at a reaction temperature that is at least the melting point of the first compound.

16. The method according to any preceding claim, wherein the biopolymer is a polysaccharide and the molar ratio of the first compound to anhydrous glucose units of the biopolymer particles is from about 1 :1 to about 10:1.

17. The method according to any preceding claim, wherein 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; optionally wherein 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; or wherein the biopolymer particles are prepared by: a. a membrane emulsification of a dispersed phase into a continuous phase wherein the dispersed phase comprises the biopolymer in a solvent, andwherein 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.

18. A method for preparing functionalised biopolymer particles from a solvent dispersion of biopolymer particles, wherein the solvent of the dispersion is aqueous and wherein the biopolymer particles are in the form of a hydrogel, said method comprising: optionally subjecting the solvent dispersion of biopolymer particles to a solvent exchange; optionally filtering the biopolymer particles; drying the biopolymer particles to remove the solvent; mixing the dried biopolymer particles with a first compound, wherein the first compound comprises at least one carboxyl group or is an acid anhydride, and reacting the biopolymer particles with the first compound via an esterification reaction to form the functionalised biopolymer particles.

19. A method for immobilising enzymes on functionalised biopolymer particles, wherein the method comprises preparing functionalised particles by the method of any one of claims 1 to 18, and contacting the functionalised particles with an enzyme, preferably wherein the enzyme is a lipase, an oxidoreductase, or an aminotransferase.

20. The method according to claim 19, wherein the enzyme is covalently immobilised on the particles.

21. Functionalised biopolymer particles prepared by the method according to any preceding claim, preferably wherein the particles are approximately spherical and / or have a diameter from about 1 pm to about 3 mm.

22. Use of the functionalised biopolymer particles according to claim 21 for immobilising an enzyme, preferably wherein the enzyme is a lipase, an oxidoreductase, or an aminotransferase.

23. A method for performing an enzyme-catalysed reaction, the method comprising forming a reaction mixture comprising the enzyme immobilised functionalised biopolymer particles prepared by the method according to claim 19 or claim 20, and at least one substrate.

24. Use of enzyme immobilised functionalised biopolymer particles prepared by the method according to claim 19 or claim 20 for catalysing a reaction.

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