Field of the Invention

The present invention relates to a macromolecule scaffold comprising wood-derived cellulose. The present invention further relates to a method of producing a macromolecule scaffold, a kit for the production of a macromolecule scaffold and a kit comprising a macromolecule scaffold and a macromolecule.

Background to the Invention

The use of recombinant enzymes as catalysts for chemical reactions is increasing in application due to their relatively high selectivity and green credentials in comparison to traditional synthesis routes. Issues such as relatively high cost, poor recyclability and poor stability can, however, make enzyme-based catalysis unviable for some industrial processes. These issues can be mitigated through enzyme engineering as well as the immobilisation of enzymes onto inert solid supports which can enhance their physico chemical stability (allowing them to tolerate harsher reaction conditions) and allow for facile recovery and re-use from batch reactors. Immobilisation of enzymes onto porous supports or packed particle beds also permits their use as de-facto heterogenous catalysts for continuous flow biocatalysis (CFB) (see Figure 1a). During CFB, reactants are passed over an enzyme-immobilised porous support to produce a continuous product stream which can then be further processed or recovered. However, drawbacks associated with CFB include relatively high cost and non-reusability of some scaffold materials, and loss of enzyme activity over time.

The use of porous supports, or scaffolds, for immobilisation of enzymes has advantages compared to the use of packed particle beds, which are often associated with low selectivity and uncontrolled fluid dynamics in the reactor. The use of porous supports offers a high inner surface area advantageous for catalysis. Furthermore, aligned porous supports hold certain advantages over non-aligned equivalents, including relatively low back-pressures needed to pass fluids over the active surface (a beneficial property for chromatography and other flow-based applications including CFB). Aligned macroporous materials can be produced by a number of methods including, for example, directional freezing and freeze-drying technique, or other complex synthetic processes which are resource-intensive. Global biomass growth generates approximately 1.5 trillion tonnes of cellulose annually, making it a practically inexhaustible renewable resource. In addition to hyperabundance, plant-derived cellulose is cheap, biodegradable, non-toxic and inert. Cellulose may be extracted from wood, which has a natural anatomy of channels aligned in an axial direction, to efficiently transport water-based solutions in the plant. However, wood-derived cellulose is commonly extracted as a pulp in which the native aligned porous structure of the original wood is lost. The process of extracting wood- derived cellulose in this way is usually via the Kraft process, i.e. the base-catalysed hydrolysis of lignin and hemicellulose (the other major components of wood).

The development of cost-effective, green and effective enzyme immobilisation techniques will help to facilitate the adoption of CFB by industry and academia. In addition to the development of improved protein immobilisation techniques and improved scaffolds for use in CFB, for example, there is a need for improved scaffolds for other macromolecules. Macromolecules other than proteins, for example nanobodies, antibodies, DNA, RNA and lectins, are often required to be immobilised on solid supports. Such immobilised macromolecules can be used, for example, for ligand capture in flow matrices, detection of toxic compounds, controlled release of therapeutic agents or the detection of specific biomolecules such as antibodies, metabolic products, or specific DNA or RNA sequences. It would be beneficial if improved scaffolds for immobilisation of macromolecules were available. It is an object of the present invention to obviate or mitigate one or more of the abovementioned problems.

Summary of the Invention The present invention relates to a macromolecule scaffold comprising wood-derived cellulose and is based, in part, on studies by the inventors in which they have shown that by utilising a relatively mild Kraft process, wood can be di-lignified without disruption of its native porous structure, resulting in aligned macroporous cellulosic materials which can be effectively used as macromolecule scaffolds. According to a first aspect of the present invention there is provided a macromolecule scaffold comprising wood-derived cellulose wherein the cellulose comprises a plurality of elongate lumen and wherein the scaffold is substantially de-lignified.

The present inventors have surprisingly found that they can obtain a cellulose structure comprising a plurality of elongate lumen which can be effectively utilised as a macromolecule scaffold. The immobilisation of macromolecules on the inner surface of the lumen results in a macromolecule scaffold which is particularly useful in a CFB processes, for example, due to the large surface area provided.

The macromolecule scaffold of the present invention comprises wood-derived cellulose. As will be appreciated by the skilled person, cellulose is a polysaccharide consisting of a linear chain of b(1 4) linked D-glucose units. Cellulose is a structural component of the cell wall of plants in addition to certain bacteria and algae, for example. As will be appreciated by the skilled person, the phrase “wood-derived cellulose”, as used herein, means that the cellulose used to form the macromolecule scaffold of the invention has been obtained from ligneous wood rather than an alternative cellulose source such as non-ligneous plants or bacteria, for example. By developing the method described herein, the present inventors have been able to obtain a cellulose scaffold which retains the native luminal structure of the wood. The macromolecule scaffold of the invention therefore comprises a plurality of elongate lumen (also described herein as channels). These channels are particularly useful as they provide a large surface area on which a macromolecule can be immobilised, such as a protein or enzyme for catalysis.

In embodiments of the present invention, the macromolecule scaffold is derived from lengths of wood. In embodiments the macromolecule scaffold is derived from treated lengths of wood. The treatment of the wood is described in more detail below.

The term “length of wood” as used herein means a piece of wood that is substantially cuboid, tubular, cylindrical or similar and elongated along one axis, and having a luminal structure (i.e. a structure comprising channels) along said elongated axis. In embodiments, the cross-sectional size of each end of the length of wood is substantially similar. The actual length of each length of wood may depend upon the wood type used and the porous nature of each wood type, for example. The length of wood may vary depending on the application for which the macromolecule scaffold is intended. By altering the length of wood, it is possible to regulate residence time, flow rates, flow volumes and back-pressures in reactions/interactions occurring within the scaffold. For example, when the macromolecule scaffold is to be used for an enzymatic reaction which requires a longer residence time (i.e. contact time between the enzyme and substrate), a longer length of wood may be utilised. In embodiments of the present invention the length of each length of wood may be between 30 mm and 300 mm, for example. In embodiments, the length may be between 40 mm and 250 mm. In embodiments of the invention the lengths of wood may be around 50 mm in length.

The macromolecule scaffold of the present invention may be derived from any suitable wood type, as will be understood by the skilled person. For example, the macromolecule scaffold may be derived from a softwood or a hardwood. Examples of suitable softwoods include, for example, American basswood, Sitka spruce, Scots pine, Cedar, Douglas Fir and Hemlock. Examples of suitable hardwoods include, for example, European Oak, Common Ash, Red Cherry, Chestnut and English Elm. In preferred embodiments, the macromolecule scaffold is derived from a softwood. In preferred embodiments, the macromolecule scaffold is derived from American basswood since the diameter of the channels that make up this wood are relatively homogenous.

As the skilled person will appreciate, wood is essentially composed of cellulose, hemicellulose and lignin. The macromolecule scaffold of the present invention comprises cellulose which has been derived from wood. The macromolecule scaffold of the present invention is substantially de-lignified. The phrase “substantially de- lignified” as used herein, means that the majority of the lignin present in the wood from which the macromolecule scaffold is derived has been removed. However, it is possible that the macromolecule scaffold may comprise small amounts of lignin. In embodiments, the macromolecule scaffold does not comprise lignin.

Lignins are a class of complex cross-linked organic polymers which, by nature of their evolutionary design, do not react or degrade easily. This combination of relative inertness and poorly-defined structure makes the utilisation of lignin as a functional scaffold material relatively challenging compared to cellulose - which has a much more defined structure by comparison. Therefore, the removal of lignin from wood is beneficial since it leaves a scaffold of relatively pure cellulose which may be further modified through defined chemical (e.g., covalent attachment of functional groups) or physical (e.g., heat treatment to alter crystallinity) means. There also exists a class of proteinaceous cellulose-specific binding domains (termed carbohydrate binding modules, CBMs) which may be attached to macromolecules of interest to increase their affinity to a cellulose scaffold; the presence of lignin may interfere with such attachment and so its removal is beneficial.

Hemicellulose is covalently attached to lignin and thus the removal of lignin typically results in the removal of hemicellulose through the same mechanism. As such, in embodiments the macromolecule scaffold is substantially free of hemicellulose.

In some embodiments of the present invention the macromolecule scaffold may comprise cellulose type II polymorph. In such embodiments, the macromolecule scaffold may comprise cellulose substantially of type II polymorph. In embodiments of the present invention the macromolecule scaffold may comprise cellulose type Ib polymorph. In such embodiments, the macromolecule scaffold may comprise cellulose substantially of the type Ib polymorph. In embodiments the macromolecule scaffold may comprise cellulose of type II and type Ib polymorphs.

As will be appreciated by the skilled person, naturally occurring cellulose is known as cellulose type I, which exists in parallel strands without inter-sheet hydrogen bonding. Cellulose Ib is the main form of cellulose found in higher plants. Cellulose type II, on the other hand, is thermodynamically more stable and exists in antiparallel strains with inter-sheet hydrogen bonding.

In embodiments in which a protein e.g. enzyme is attached to the macromolecule scaffold using a carbohydrate binding module (CBM) (described in detail below) it is particularly advantageous if the scaffold comprises cellulose of the type II polymorph, because this has been found to result in particularly efficient immobilisation and high total macromolecule loading.

The present inventors have developed a process in which wood can be di-lignified without disruption of its native porous structure, resulting in aligned macroporous cellulosic materials which can be utilised as macromolecule scaffolds. Development and subsequent improvement of the process by the inventors has surprisingly resulted in a particularly effective macromolecule scaffold. The present invention therefore also provides a macromolecule scaffold comprising wood-derived cellulose wherein the cellulose comprises a plurality of elongate lumen and wherein the scaffold is substantially de-lignified, and wherein the scaffold is obtainable by a process comprising the following steps: a) treating wood in a solution comprising sodium hydroxide and sodium sulphite; and b) treating said wood in a solution comprising hydrogen peroxide.

In embodiments the wood comprises one or more lengths of wood. The present invention therefore also provides a macromolecule scaffold comprising wood-derived cellulose wherein the cellulose comprises a plurality of elongate lumen and wherein the scaffold is substantially de-lignified, and wherein the scaffold is obtainable by a process comprising the following steps: a) treating one or more lengths of wood in a solution comprising sodium hydroxide and sodium sulphite; and b) treating the one or more lengths of wood in a solution comprising hydrogen peroxide.

The term “solution” as used herein is used to describe any liquid composition in which the components are substantially dissolved and/or are suitably mixed. In preferred embodiments, the solution comprises an aqueous solution i.e. a solution in which the components are dissolved in, or mixed in, water. In embodiments of the present invention the sodium hydroxide and sodium sulphite are dissolved in water. In embodiments of the present invention the hydrogen peroxide is dissolved in water.

The term “treating” or “treated” as used herein means that wood e.g. lengths of wood are contacted with and/or immersed in a solution comprising sodium hydroxide and sodium sulphite or hydrogen peroxide. In embodiments, the scaffold is obtainable by a process comprising treating the wood by reflux. The scaffold is therefore obtainable by a process comprising the following steps: a) treating one or more lengths of wood by reflux in a solution comprising sodium hydroxide and sodium sulphite; and b) treating the one or more lengths of wood by reflux in a solution comprising hydrogen peroxide.

The skilled person will understand the term “reflux” as used herein. However, for the avoidance of doubt, this term is used to describe a technique in which a solution is heated to produce vapours, which are then condensed and returned to the solution.

The process of heating and condensing may be repeated any number of times during the course of each stage of the treatment. “Reflux conditions” refers to reaction conditions suitable to enable reflux to occur. It will be apparent to the skilled person what these reaction conditions may be for a given treatment solution.

The method used to obtain the macromolecule scaffold of the invention comprises treating wood in a solution comprising sodium hydroxide and sodium sulphite.

In embodiments of the present invention, the method comprises adding the wood to the solution comprising sodium hydroxide and sodium sulphite.

In embodiments, the sodium hydroxide is present in the solution at between around 2 to 10 M and/or the sodium sulphite is present in the solution at around 0.1 to 1.2 M. In preferred embodiments the sodium sulphite is present in the solution at around 0.4 M. In preferred embodiments the sodium hydroxide is present in the solution at around 7.5 M. The present inventors have surprisingly found that such conditions result in the scaffold substantially comprising cellulose of the type II polymorph. In embodiments in which a protein e.g. enzyme is attached to the macromolecule scaffold using a carbohydrate binding module (CBM) (described in detail below) it is particularly advantageous if the scaffold comprises cellulose of the type II polymorph, because this has been found to result in particularly efficient immobilisation and high total protein loading. In alternative embodiments the sodium hydroxide is present in the solution at around 2.5 M. The present inventors have surprisingly found that such conditions result in the scaffold substantially comprising cellulose of the type Ib polymorph. A cellulose 1b polymorph may be advantageous under certain circumstances, for instance certain forms of chemical functionalisation may proceed more efficiently with a cellulose 1b polymorph due to beneficial steric effects (i.e., a favourable conformation promoting reactivity at selected positions). The present inventors have surprisingly found that by modifying the sodium hydroxide concentration, the cellulose polymorph can be “tuned”. This surprising finding allows the user to select the cellulose polymorph based on the end use.

In embodiments, the wood is treated by reflux in a solution comprising sodium hydroxide and sodium sulphite at around 140°C for around 16 hours. In embodiments, the reflux conditions comprise agitation.

In embodiments of the present invention, the hydrogen peroxide is present in the solution at between about 5% and about 20%. Preferably the hydrogen peroxide is present in the solution at greater than about 5%, preferably greater than about 10%, preferably greater than about 15%. Preferably the hydrogen peroxide is present in the solution at about 20%. Through detailed experimentation, the inventors have found that by having a hydrogen peroxide concentration between about 5% and about 20%, preferably around 20%, macromolecule loading and immobilisation efficiency to the resulting scaffold is improved. Surprisingly, by obtaining the macromolecule scaffold of the present invention using the method disclosed herein and making use of between 5% and 20% hydrogen peroxide, the macromolecule scaffolds demonstrate much improved macromolecule loading characteristics. Unless otherwise stated, the concentration of hydrogen peroxide is the percent by volume in the solution.

In embodiments, the wood is treated by reflux in a solution comprising hydrogen peroxide at around 125°C for around 2 hours.

In embodiments, steps a) and b) are carried out in the order a) and then b). In embodiments of the present invention, the method further comprises step a1), between steps a) and b), step a1) comprising: washing the wood with water. Washing the wood with water removes a significant proportion of the hydrolysed lignin from the scaffolds. In embodiments, step a1) may further comprise treating the wood by reflux in water. Suitable reflux conditions are around 125°C for around 1 hour.

In embodiments, the method further comprises step c), following step b), step c) comprising: treating the wood with a composition comprising absolute alcohol.

The macromolecule scaffolds of the present invention may be washed in water before storage. The macromolecule scaffolds may be stored in alcohol, for example, preferably around 5-40% alcohol, 10-30% alcohol, 15-25% alcohol or 20% alcohol, until use.

Before use, a buffer solution may be flushed through the macromolecule scaffold in order to remove any residual alcohol. The buffer used will be dependent on the intended application of the macromolecule scaffold and the desired pH of the macromolecule scaffold. The pH can have a result on the macromolecule/protein/enzyme uptake of the resultant scaffold, the stability of the immobilised macromolecule, the activity of an immobilised macromolecule (e.g. enzyme), or the activity of a chemical reaction intended to take place in or on the scaffold. Where a pH of between 7.1 and 9.1 is required, the buffer solution may comprise TRIS. Preferably the TRIS is present in the buffer solution between about 50 mM and 150 mM and the potassium chloride is present between about 50 mM and about 200 mM. More preferably the TRIS is present in the buffer solution at about 150 mM and the potassium chloride is present at about 50 mM. Preferably the buffer has a pH of between about 7.1 and about 9.1. More preferably the buffer has a pH of about 7.1. Where a pH of between 5.8 and 8 is required, the buffer solution may comprise sodium phosphate. Where a pH of between 3.6 and 5.6 is required, the buffer solution may comprise sodium acetate.

The buffer may further comprise a salt, for example sodium chloride, magnesium chloride, calcium chloride, sodium sulphate or ammonium sulphate. Preferably the salt comprises potassium chloride. Preferably the salt is present in the buffer solution between about 10 mM and 300 mM. It has been found that the use of salt in the buffer stabilises the macromolecules immobilised to the scaffold, for example proteins.

As set out above, the macromolecule scaffold of the present invention may be derived from any suitable wood type, as will be understood by the skilled person. For example, the wood may be a softwood or a hardwood. Examples of suitable softwoods include, for example, American basswood, Sitka spruce, Scots pine, Cedar, Douglas Fir and Hemlock. Examples of suitable hardwoods include, for example, European Oak, Common Ash, Red Cherry, Chestnut and English Elm. In preferred embodiments, the wood is a softwood. In preferred embodiments, the wood is American basswood since the diameter of the channels that make up this wood are relatively homogenous compared to other types of wood.

The term “macromolecule”, as used herein, refers to a large molecule comprising many atoms and includes for example, proteins, nucleic acids (for example DNA and RNA), lectins, carbohydrates, lipids, antibodies and nanobodies.

In embodiments of the present invention, the macromolecule scaffold is a protein scaffold, more preferably an enzyme scaffold. The inventors have shown that the scaffold of the present invention is particularly useful in CFB reactions in which an enzyme is immobilised on the scaffold.

In embodiments of the present invention, the macromolecule scaffold comprises a macromolecule bound to the macromolecule scaffold. The macromolecule scaffolds of the invention can be used with any macromolecule of interest. The macromolecule may comprise a protein, nucleic acid (for example DNA or RNA), lectin, carbohydrate, lipid, antibody or nanobody, for example. In preferred embodiments, the macromolecule is a protein.

In embodiments of the present invention the protein may comprise an unmodified protein or an engineered protein. In embodiments the protein may comprise an enzyme. As will be appreciated by the skilled person, an enzyme is a protein that acts as a biological catalyst. The macromolecule scaffolds of the invention can be used with any protein of interest. For example, in embodiments in which the protein is an enzyme, the enzyme may comprise a hydrolase, isomerase, lyase, oxidoreductase, synthetase or transferase. The enzymes may be cofactor dependent or cofactor independent. The macromolecule scaffolds of the present invention are particularly useful in CFB reactions, for example. Any enzyme required to catalyse a reaction of interest in such a CFB system may be bound to the macromolecule scaffold of the present invention. In embodiments of the present invention the macromolecule may comprise a carbohydrate binding module (CBM). The CBM may be native to the macromolecule or the macromolecule may be engineered to comprise a CBM.

In embodiments of the present invention the CBM may be attached to the macromolecule at either the N-terminal domain or the C-terminal domain. The inventors found no substantial difference in immobilisation performance when the macromolecule was attached via the N- or C- terminal domain.

CBMs, previously termed cellulose binding domains (CBDs), are a class of non-catalytic protein domain which have exceptional affinity binding properties to cellulose and other carbohydrates. Often found as a sub-domain of carbohydrate-active enzymes such as cellulases or xylanases, CBMs have a wide variety of sequences and structures (60+ families of CBMs currently known) which can recognise and bind to different forms of cellulose and other carbohydrates.

The skilled person will appreciate that any suitable CBM may be used. The CBM may comprise a Type A or Type B CBM. The CBM may comprise one of the following CBMs: CBM1 , CBM2a, CBM3a, CBM28 or CBM30, for example.

In embodiments in which the macromolecule comprises a CBM, the macromolecule may be bound to the macromolecule scaffold via the CBM. In such embodiments, the CBM binds to the cellulose of the macromolecule scaffold, thereby attaching the macromolecule to the scaffold.

By engineering CBMs onto the termini of recombinant proteins, the macromolecule scaffolds of the invention can be employed as low-cost, renewable and biodegradable porous monoliths for enzyme immobilisation without any further chemical functionalisation.

In alternative embodiments of the present invention the macromolecule may not contain a CBM. In such embodiments the macromolecule may be bound to the macromolecule scaffold by covalent coupling, for example. Various chemical coupling approaches exist for the attachment of macromolecules, for example proteins, to cellulose matrices which could be applied to the present invention. Suitable approaches include, for example carbodiimide chemistry, silane-epoxide bonding, and amine coupling with aldehyde or carboxylic acid groups introduced to the cellulose surface, among others.

In embodiments, the macromolecule may be bound to the scaffold within the elongate lumen. The macromolecule may also be bound to any internal structure within the macromolecule scaffold.

The term “within” as used herein is intended to mean that the macromolecule is bound to an inner surface of the lumen or an inner surface of the macromolecule scaffold. The immobilisation of the macromolecule on the inner surface of the lumen results in a macromolecule scaffold which is particularly useful in a CFB process, for example, due to the large surface area provided.

In an embodiment of the present invention a solution comprising the macromolecule to be bound is flushed through the macromolecule scaffold in order to immobilise the macromolecule. The present invention therefore also provides a method of immobilising a macromolecule on a macromolecule scaffold, the method comprising flushing a macromolecule solution through the scaffold. The macromolecule scaffold may be the macromolecule scaffold of the present invention, as described herein. Preferably the macromolecule solution is flushed through at a rate of between about 5 pi min ¹ and 100 mI min ^-1 per square centimetre of scaffold cross-sectional area. Even more preferably the macromolecule is flushed through at a rate of about 5 mI min ¹ per square centimetre of scaffold cross-sectional area.

In an embodiment of the present invention the concentration of the macromolecule in the macromolecule solution may be between about 0.5 mg ml ¹ to about 10 mg ml ¹ . Preferably the concentration of the macromolecule in the macromolecule solution is about 10 mg ml ¹ .

The present inventors have surprisingly found that by immobilising macromolecule at the flow rate and concentrations described above, immobilisation yield and total macromolecule loading can be optimised.

The present invention also provides a method of producing a macromolecule scaffold the method comprising the following steps: a) treating wood in a solution comprising sodium hydroxide and sodium sulphite; and b) treating said wood in a solution comprising hydrogen peroxide.

Features of the method are described above in relation to the macromolecule scaffold obtainable by a method.

In embodiments the wood may comprise one or more lengths of wood. As such, the present invention also provides a method of producing a macromolecule scaffold the method comprising the following steps: a) treating one or more lengths of wood in a solution comprising sodium hydroxide and sodium sulphite; and b) treating the one or more lengths of wood in a solution comprising hydrogen peroxide.

In embodiments the macromolecule scaffold comprises wood-derived cellulose comprising a plurality of elongate lumen and wherein the scaffold is substantially de- lignified. The macromolecule scaffold may be macromolecule scaffold of the first aspect of the present invention, as described above.

The present invention also provides for the use of the macromolecule scaffold of the invention in a process catalysed by an enzyme bound to the macromolecule scaffold.

The present invention also provides the use of the macromolecule scaffold of the invention in a continuous flow biocatalysis process.

The present invention also provides the use of the macromolecule scaffold of the invention in the treatment of wastewater. For example, immobilised lipases and proteases can be used for the treatment of wastewater. Immobilised laccase can also be used remove small quantities of high impact substances such as dyes and pharmaceuticals. As mentioned above, the immobilisation of macromolecules on the inner surface of the lumen results in a macromolecule scaffold which is particularly useful in enzymatic reactions catalysed by said macromolecule, due to the large surface area provided by the inner surface of the lumen. The scaffolds of the present invention can therefore be effectively employed in CFB processes, the treatment of wastewater, lateral flow clinical diagnostic equipment, processing or quality control in the food and beverage industries, etc.

Advantageously, the scaffolds of the present invention may be reused any number of times in the process catalysed by the macromolecule bound to the scaffold. Reuse of the macromolecule scaffold may involve removal of the scaffold from the reaction buffer or replacement of the reaction buffer.

The present invention also provides a kit for the production of a macromolecule scaffold comprising wood, sodium hydroxide, sodium sulphite and hydrogen peroxide. Preferably the wood comprises lengths of wood.

The present invention also provides a kit comprising a macromolecule scaffold according to the invention and a macromolecule, preferably a protein, more preferably an enzyme.

The present invention yet further provides a kit comprising a macromolecule scaffold according to the invention and a flexible tube. The flexible tube may comprise a polymer. The flexible polymer tube may comprise any chemical resistant polymer. In embodiments the chemical resistant polymer comprises PVC.

The present invention also provides a kit comprising a macromolecule scaffold according to the present invention and a flexible polymer tube wherein the macromolecule scaffold is contained within the flexible polymer tube.

The described and illustrated embodiments are to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the scope of the inventions as defined in the claims are desired to be protected. The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention as set out herein are also to be read as applicable to any other aspect or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each exemplary embodiment of the invention as interchangeable and combinable between different exemplary embodiments. It should be understood that while the use of words such as “preferable”, “preferably”, “preferred” or “more preferred” in the description suggest that a feature so described may be desirable, it may nevertheless not be necessary and embodiments lacking such a feature may be contemplated as within the scope of the invention as defined in the appended claims. In relation to the claims, it is intended that when words such as “a,” “an,” or “at least one,” are used to preface a feature there is no intention to limit the claim to only one such feature unless specifically stated to the contrary in the claim.

Detailed Description of the Invention The present invention will now be further described with reference to the following figures which show:

Figure 1 : a) Schematic overview of a typical packed-bed CFB process b) Scheme depicting typical macromolecule scaffold synthesis c) Scheme depicting the immobilisation of a CBM-enzyme construct to a cellulose surface.

Figure 2: Characterisation of CBM-Reporter Constructs: a) Schematic of the six CBM- reporter constructs b) Photographs of purified proteins under (i) white light and (ii) UV (from left to right, mECFP-CBM2A, CBM2a-mEGFP, mCitrine-CBMI, CBM30- mOrange, CBM3A-mCherry and mNeptune-CBM28. c) Graph showing the UV-vis spectra of the purified proteins.

Figure 3: Photographs of the lengths of wood/cellulose scaffolds at each point in the initial synthesis protocol a) - e) Schematic depicting the initial scaffold synthesis protocol f) Basswood portion (left) and processed scaffolds (right) for comparison. Figure 4: Photographs showing: a) a representative scaffold inserted into 80 mm length PVC tubing, and b) an image of syringe-tubing connection via Luer-lock attachments.

Figure 5: a) Photographs of CBM-fluorescent proteins (CBM-FP) purified from expression trials (prior to dilution to 5 ml): 1. mCitrine-CBM1 , 2. CBM30-mOrange, 3. CBM3A-mCherry, 4. mNeptune-CBM28, 5. mECFP-CBM2A and 6. CBM2a-mEGFP. b) Photograph of a 96-well plate of the CBM-FP solutions before and after scaffold flowing through (FT), and after a subsequent 1.5 ml FT of buffer, under ordinary light (left) and a black light (right) c) Graphs showing the UV-vis spectrophotometry traces of the CBM-FPs before (left) and after (centre) FT, and after a further 1.5 ml FT of buffer (right) d) Visible light (above) and black light (below) photographs of the cut-up scaffolds after FT and 1.5 ml buffer flush.

Figure 6: Visible light photographs of scaffolds soaked in acetone a) before and b) after drying. Numbers 1 - 14 represent the scaffold synthesis conditions.

Figure 7: Cross-sectional Field Emission Gun Scanning Electron Microscopy (FEG- SEM) images of the scaffolds displaying aligned microporosity. Representative examples of synthesis conditions a) 1, b) 5, c) 6, and d) 8. Scale bars = 50 pm.

Figure 8: Graph showing the Wide-angle X-ray diffraction (WAXD) patterns of the scaffolds produced under various conditions listed. Dashed, dotted and solid lines represent 7.5, 5 and 2.5 M NaOH concentrations during the Kraft process, respectively.

Figure 9: Visible light photographs of scaffolds produced under the Definitive Screening Design (DSD) synthesis conditions after flowing-through CBM3-mCherry solution and having been subsequently cut up.

Figure 10: Graphs showing the a) loading and b) immobilisation efficiency response surface model (RSM) prediction profilers for the scaffold synthesis DSD. Dashed lines represent confidence intervals and dotted lines indicate prediction profiler settings. Graphs showing the c) loading and d) immobilisation efficiency RSM models calculated from experiments 1 - 14 (dots) and validated with experiments 15 - 18 (diamonds).

Figure 11 : Graphs showing the a) immobilisation efficiency and b) loading RSM prediction profilers for the flow conditions optimisation DSD. Dashed lines represent confidence intervals and dotted lines indicate prediction profiler settings. Graphs showing the c) immobilisation efficiency and d) loading RSM models calculated from experiments 1 - 13 (dots) and validated with experiment 14 (cross).

Figure 12: Graph showing the results of the flow conditions optimisation DOE experiments 14, 15 and 16 with increasing buffer concentration: immobilisation yield (dots) and loading (triangles).

Figure 13: Graphs showing the a) loading and b) immobilisation efficiency of the CBM- FPs loaded at ~10 mg ml-1 before and after a 10 ml buffer flush. Graphs showing the c) loading and d) immobilisation efficiency of CBM-mEGFP and CBM-mNeptune loaded at 5 mg ml-1 before and after a 10 ml flush (extends to 80 ml for CBM-mEGFP). Photographs of the e) CBM-mEGFP solution before and after initial loading f) Visible light (above) and black light (below) photographs of CBM-FPs loaded onto scaffolds (after 10 ml buffer flush).

Figure 14: Diagram of the reaction scheme depicting the conversion of (S)-a- Methylbenzylamine (MBA) into acetophenone via the scaffold immobilised CBM2a-ooTA enzyme.

Figure 15: Graphs showing the a) activity of free CBM2a-ooTA in solution determined though acetophenone assay (absorbance at 245 nm) and the b) activity of scaffold- immobilised CBM2a-ooTA relative to activity of free enzyme in solution.

Figure 16: Photographs showing visible light images of a) Basswood (left) and Oak (right) derived scaffolds b) images of both scaffolds before (left) and after (right) immobilisation of CBM3a-mCherry.

Figure 17: Photographs showing visible light images of the scaffolds after having cell lysate containing mCherry (above) and CBM3a-mCherry (below) flowed through.

Figure 18: Reaction scheme showing the functionalisation of cellulose with aldehyde groups followed by covalent enzyme attachment via terminal amine groups and subsequent reduction into amine bonds. Figure 19: a) A graph showing the UV-Vis absorbance profile with absorbance at 400 nm associated with the hydrolysis of 4-Nitrophenyl palmitate (NPP) after passing a 10 mM solution in hexane through a lipase-immobilised scaffold (lipase-free control also included) b) A visible light photograph of a scaffold with covalently immobilised lipase and a lipase-free control scaffold after passing through a 10 mM solution of NPP in hexane c) A graph showing the UV-Vis absorbance profile with absorbance at 400 nm associated with the hydrolysis of NPP for a suspension of lipase in a 10 mM solution of NPP in hexane d) A graph showing the percentage conversion of NPP to hydrolysis products over time for a 10 mM NPP solution in hexane with lipase-immobilised scaffold and a lipase-free CS control.

Examples

The inventors carried out a number of experiments in which aligned macroporous cellulose scaffolds (CSs) were produced through the de-lignification of wood via a relatively mild Kraft process (Figure 1b). Recombinant proteins were then immobilised to the internal surfaces of the scaffolds via CBM-cellulose affinity binding (Figure 1c) or covalent bonding. By identifying the more significant process parameters, the present inventors have surprisingly been able to maximise total protein loading and immobilisation efficiency, achieving maximum values of 5.24 wt.% and 97.1%, respectively. Six different CBMs were initially investigated, and fluorescent proteins were initially employed as reporter tags to facilitate characterisation and optimisation, and to determine if any localised binding was occurring on the scaffolds. Finally, a construct based on an w-transaminase (wTA) from Bacillus megaterium and the best performing CBM (namely, CcCBM2a from C. cellulovorans - EngD) was produced, immobilised onto the CSs, and tested in a CFB setup. The immobilised enzyme displayed c.a. 95 ± 5% conversion efficiency relative to the free enzyme in solution under analogous conditions, demonstrating that CS immobilised CBM-tagged enzymes can be successfully employed for CFB.

Example 1 : A range of Carbohydrate Binding Modules (CBMs), linkers and attached proteins can be employed to immobilise proteins to CSs.

CBMs can be grouped into three types, namely surface-binding CBMs (type A), glycan chain-binding (type B) and small sugar-binding (type C). Six CBMs from various organisms were investigated by the present inventors. Four of these belong to the Type A class of CBMs including CBM1 from T. reesei cellobiohydrolase Cel7A, CBM2a from A. cellulolyticus GH5 endoglucanase, CBM2a from C. cellulovorans EngD, and CBM3a from C. thermocellum CipA scaffolding. The other two CBMs investigated, CBM28from Bacillus sp.1139 Cel5a endoglucanase and CBM30 from C. thermocellum Endoglucanase CelJ belong to the Type B class of CBMs.

For easy visualisation and quantification of CBM binding, the six different CBMs were fused to six different fluorescent proteins. Variants of fluorescent proteins were selected that were reported to be monomeric, these included mECFP, mEGFP, mCitrine, mOrange, mCherry and mNeptune. Three of the CBMs were attached to the N-terminal end of the fluorescent protein whilst the other three were attached to the C-terminus. All CBM-Fluorescent protein fusions were linked either by a glycine/serine rich linker or a threonine/proline rich linker derived from T. reesei Cel7A (TrCel6A). A summary of the CBM-FP constructs’ design is given in Table 1 below and Figure 2A. All six constructs were expressed in E. coli and purified by Ni2+ immobilised metal ion affinity (IMAC) as confirmed by UV-vis spectrophotometry (Figure 2C).

Table 1. Summary of the design of the CBM-FP constructs. Recombinant protein design, expression and purification method

CBM-FP constructs - Construct design and cloning:

Genes encoding the fusion proteins were codon optimised for expression in E. coli using PartsGenie and synthesised (Twist Bioscience). For protein expression the genes encoding the fusion protein were cloned into expression vector pBbAlc. The genes of interested were amplified by PCR using CloneAmp polymerase premix (ClonTech) and the primers shown below:

The template was removed by Dpnl (New England Biolabs) digest and the DNA purified using a Qiaquick PCR purification kit (Qiagen). The PCR products were cloned into Ndel/Xhol (New England Biolabs) linearised pBbMc using Infusion HD (Clontech). DNA was transformed into NEB5alpha cells (New England Biolabs).

Protein expression and purification:

An overnight preculture was grown in LB medium supplemented with 34 pg/ml chloramphenicol at 37°C. 500 ml phosphate buffered terrific broth (Formedium) supplemented with 34 pg/ml chloramphenicol at 37°C was inoculated with 5 ml pre culture and grown at 37°C. Once the culture had reached an Oϋboo of 0.5-1 cultures were induced with 1 mM IPTG and grown at 37°C overnight. In the case of the CBM2a- mEGFP construct protein solubility was increased by dropping the incubation temperature to 25°C post-induction. The following morning cells were harvested by centrifugation and stored at -20°C until required. Cells were resuspended in buffer A (200 mM KCI, 50 mM Tris pH 7.5) supplemented with protease inhibitor cocktail and DNAase (Sigma) and lysed by cell disruption at 20 KPsi. Lysate was clarified by ultracentrifugation at 14,000 g for 1 hour and the supernatant applied to a Ni-NTA agarose column (Qiagen). The column was washed with 5 column volumes of buffer A supplemented with 10 mM imidazole and then 40 mM imidazole. The protein was eluted with buffer A supplemented with 250 mM imidazole. If required, the purified protein was concentrated in a 10k MWCO Vivaspin20 centrifugal device before being desalted into buffer A using a desalting column in order to remove the imidazole.

Protein concentration was quantified using a Cary 60 UV-vis spectrophotometer (Agilent). Extinction coefficients are detailed in Table 2 below. Values for A280 were calculated from the amino acid sequence using the Protparam tool on the ExPASy proteomics server (https://web.expasy.org/protparam/) whilst extinction coefficients for the chromophores were taken from the fluorescent protein database (https://www.fpbase.org).

Table 2: UV-vis parameters used for protein quantification Cellulose scaffold synthesis method

Initial synthesis protocol:

A block of Tilia Americana basswood (Stockport Hobbycraft Store, UK) was sawn into 10 x 10 x 50 mm portions with the grain of the wood following the long (50 mm) axis

(Figure 3). Each wood portion weighed approximately 0.9 - 1 g and was gently sanded by hand with abrasive paper to remove any residual wood flakes. Separately, 50 g NaOH and 25.2 g Na2SC>3 were dissolved in Dl water to a final volume of 500 ml (2.5 M NaOH, 0.4 M Na2SC>3). 25 portions of wood were added to a 1 L round-bottomed flask before addition of the 500 ml NaOH/Na2SC>3 solution. The mixture was then refluxed at 140°C for 16 hours with gentle magnetic stirring. The solution was then drained and the wood portions were washed with cold Dl water 3 x times, before being refluxed with Dl water (500 ml) at 125°C for 1 hour a single time. The scaffolds were then refluxed in 500 ml H2O2 (5 %, Sigma UK) for 2 hours at 125°C along with c.a. 5 drops of antifoam- 204 (Sigma-Aldrich) (caution: significant foaming can still occur even with antifoaming agent). This H2O2 treatment was repeated two further times. The scaffolds were then washed in cold Dl water 3 x times before being placed in 200 ml absolute ethanol overnight. Dl water was then added to bring the final volume to 1 L (20% v/v EtOH) where the scaffolds were then stored at room temperature (RT) until use.

Immobilisation procedure

Initial immobilisation procedure:

The initial immobilisation procedure, used to investigate the different CBMs and to optimise the scaffold synthesis was performed as follows:

Transparent PVC tubing (inner diameter 8 mm, outer diameter 12 mm) was cut to a length of approximately 80 mm. A scaffold would then be trimmed slightly with a razor blade into a cylindrical shape (initially cuboidal) and inserted into the tubing ensuring a tight fit and sitting in the centre of the tubing (Figure 4a). A 50 ml plastic Luer Lock syringe was connected to one end of the PVC tubing using a 3-way Luer Lock valve (for loading/discharging solutions), male/female Luer Lock attachments and poly(vinylidene fluoride) (PVDF) tubing (Figure 4b). 30 ml of buffer solution (50 mM TRIS, 200 mM KCI, pH 7.5) was then flushed through the scaffold at a rate of 250 pi min ^-1 to flush out residual EtOH. 5 ml of CBM3A-mCherry (2 mg ml ¹ ) was then flushed through the scaffold at a rate of 100 mI min ¹ (Aladin syringe pump, World Precision Instruments) and collected. A further 2 ml of buffer was flushed through and collected to wash out non-adsorbed CBM3-mCherry. The CBM3-mCherry concentration before (c.a. 2 mg ml ¹ ) and after passing though the scaffold (including 2 ml additional flush) was measured via UV-visible spectrophotometry (Abs [587], £ = 72000 M-1 cm-1 , Mw = 48892.28 g mol ¹ ). The experiments were conducted at room temperature (RT, typically 19 ±3°C).

Results After synthesis, the CBM-FP constructs were flowed through scaffolds and their concentration was determined before and after flow-through via UV-visible spectrophotometry (Figure 5). This showed binding of all constructs to the scaffolds, which was additionally confirmed via visible light images under a black light (Figure 5d).

Example 2: The effect of Cellulose Scaffold (CS) synthesis conditions on their resultant physical properties - increasing total protein loading and immobilisation efficiency

The treatment of cellulosic materials (e.g. cotton) with concentrated aqueous sodium hydroxide (NaOH) is known to increase their capacity to adsorb substances (e.g. dyes) in a process historically known as Mercerisation. This is attributed to a change from the naturally occurring cellulose polymorph Cellulose Ib to the Cellulose II polymorph. Here, the inventors increased the concentration of NaOH during the synthesis of CSs to determine whether this would result in a Cellulose II type structure, which could potentially be exploited to improve absorptive capacity of the scaffolds (i.e. increase total protein loading and immobilisation efficiency). Other factors involved in the CS synthesis were also varied - namely Na ₂ S0 ₃ concentration, total Kraft solution volume, H2O2 concentration and H2O2 volume, in addition to NaOH concentration. This was done through a Design of Experiments (DOE) Definitive Screening Design (DSD) with a summary of the experimental conditions given in Table 3 below.

Table 3: Summary s the input factors for the CS synthesis DSD Observations and Field Emission Gun Scanning Electron Microscopy (FEG-SEM)

After synthesis of the CSs, it was observed that different CS synthesis conditions resulted in a range of colours from white to light brown (Figure 6). Upon drying, some CSs underwent significant shrinkage (e.g. condition 1, 8, 9 and 12) whilst others remained relatively robust (e.g. condition 2, 7 and 14). This appeared to be correlated to the concentration of NaOH during the process, where a high NaOH concentration tended to result in greater shrinkage. FEG-SEM images from cross sections of the CSs revealed the aligned macropores of wood (xylem and phloem) were still intact following treatment (Figure 7). The porosity of the CSs was determined though wet/dry mass analysis, with porosity varying between ca. 69 - 78 % which was again correlated with NaOH concentration.

Wide-angle X-ray diffraction (WAXD)

Wide-angle X-ray diffraction (WAXD) was performed using a PANalytical X’Pert Pro instrument with a copper Ka radiation source (1.54 A wavelength), diffraction angle of 5 -60° and scanning rate of 1 ° min ¹ . Field emission gun scanning electron microscopy (FEG-SEM) was performed using an FEI Quanta 250 instrument with a working distance of 10 mm and accelerating voltage of 10 kV. Acetone-soaked CS samples were air dried overnight before being cut into small portions and adhered to 1 cm ² Al stubs with double-sided conductive carbon tape. Samples were sputter coated with 10 nm of an Au/Pd alloy prior to imaging to enhance sample conductivity, and a small amount of Ag paint was also used to improve electrical contact between the sample and carbon tape. UV-visible spectrophotometry was performed using a either a Cary 60 UV-vis spectrophotometer (protein expression and quantification) or a Clariostar Plus plate reader.

Wide-angle X-ray diffraction (WAXD) was performed on the synthesised CSs to probe any relation between microstructure and synthesis conditions. Natural wood-derived cellulose exists as highly stable crystalline cellulose Ib - but can be converted to the more thermodynamically favourable polymorph cellulose II though swelling and relaxation by treatment with concentrated NaOH. The WAXD patterns show that CSs prepared with a relatively low NaOH concentration (2.5 M) maintain the characteristic 200 and 110 Bragg reflections associated with cellulose Ib, as does the untreated Basswood (Figure 8). However, the synthesis conditions employing the relatively high NaOH concentration (7.5 M) all had an additional peak at ca. 19.5° and a slightly down shifted main peak at c.a. 21.5° - which was attributed to 110 and 020 Bragg reflections of a cellulose II type structure (Figure 8). This demonstrates that the physical properties, including the microstructure, of the CSs can be varied by changing the initial synthesis conditions.

Relationship between CS synthesis conditions and immobilisation

To determine whether a relationship between CS synthesis conditions and immobilisation of a CBM-fusion protein exists, CBM3A-mCherry solutions were flowed through the synthesised CSs and total protein loading and immobilisation efficiency was determined through UV-Vis spectrophotometry. The CSs were also cut up and visible light images taken, which showed the distribution of CBM3A-mCherry within the scaffolds (Figure 9).

Causal inferences between input variables and output factors

The previously produced data were analysed and the most significant factors from the previous screening experiments were selected to build response surface models (RSMs). The purpose of the RSMs was to allow predictions (or generate causal inferences) to be made between the significant input variables and the output factors, i.e. which input variables should be varied in order to maximise the loading and immobilisation efficiency. The RSM for loading predicted that the highest value (0.896 wt. %) would be achieved with the highest concentrations of H2O2, NaOH and Na ₂ S0 ₃ , as well as the highest Kraft solution volume (Figure 10a). The RSM for immobilisation efficiency predicted that a higher efficiency would be obtained with the highest concentrations of H2O2 and NaOH, a lower Na ₂ S0 ₃ concentration, and the highest H2O2 volume (Figure 10b). Based on these RSM predictions, further experiments (experiment no. 15 - 18, Table 4) were conducted in order to validate the models and improve the output factors to increase protein loading and protein immobilisation efficiency.

Increasing H ₂ O ₂ concentration

Experiments 16, 17 and 18 (Table 4) employed an increasing concentration of H2O2 (15, 17.5 and 20%, respectively) since there was a strong positive correlation between this factor and both loading and immobilisation efficiency. The results from these validation experiments are presented in Figures 10c and 10d. When assessing protein loading, these results correlated with previous experiments. In fact, these experiments (experiments 16, 17 and 18, Table 4) actually had a slightly higher loading than predicted by the RSM (1.01, 1.15 and 1.09 %, vs. predicted values of 0.85, 0.90 and 0.96 %) and higher than any of the previous experiments in the DSD. Similarly, the immobilisation efficiency was also improved for experiments 16, 17 and 18 shown in Table 4 (at 59.5, 67.1 and 70.5 %) compared to previous experiments - although these did not accurately follow the trend predicted by the model (RSM). Table 4 summarises the data from these experiments. The investigations undertaken by the inventors surprisingly show that the total loading and immobilisation efficiency can be substantially improved by varying the CS fabrication conditions.

Table 4: Summary of output variables and other characteristics of the CS’s produced from the CS synthesis DSD.

The optimised cellulose scaffold synthesis protocol was determined as follows:

150 g NaOH and 25.2 g Na2SC>3 were dissolved (some of the Na2SC>3 remains undissolved at RT as saturation solubility was reached) in Dl water to a final volume of 500 ml (7.5 M NaOH, 0.4 M Na ₂ S0 ₃ ). 25 portions of wood were added to a 1 L round- bottomed flask before addition of the 500 ml Na0H/Na ₂ S0 ₃ solution. The mixture was then refluxed at 140°C for 16 hours with gentle magnetic stirring. The solution was then drained and the wood portions were washed with cold Dl water 3 x times, before being refluxed with Dl water (900 ml) at 125°C for 1 hour 3 x times. The CSs were then refluxed in 875 ml H2O2 (20%) for 3 hours at 125°C a single time. Foaming was no longer an issue in this instance and no antifoam 204 was added. The CSs were then washed in cold Dl water 3 x times before being placed in 200 ml of absolute ethanol overnight. Dl water was then added to bring the final volume to 1 L (20 % v/v EtOH) where the CSs were then stored at RT until use. Example 3: Varying loading conditions to increase total protein loading and immobilisation efficiency on CSs

To increase total protein loading and immobilisation efficiency, the conditions in which the CBM-fusion constructs were immobilised onto the CSs was subject to parametric optimisation via a DSD. Here, factors that were deemed to be significant - namely pH, protein concentration, protein volume, flow rate and salt concentration - were varied. The results, presented in Table 5 below, were used to construct RSM models with the most significant factors. The RSMs predicted that the optimum conditions for immobilisation efficiency would be the lowest pH (7.1), lowest flow rate (50 mI min ¹ ) and highest protein volume (7.5 ml) (Figure 11a). Optimum conditions for loading were predicted to be the highest protein concentration and volume (the former having a greater effect), lowest flow rate (50 mI min ¹ ) and lowest pH (7.1) (Figure 11b). Based on these insights, a 14th experiment (F14) (Table 5) was performed with the intention of both validating the models and extrapolating their predictions to maximise total loading whilst maintaining a high immobilisation efficiency.

Table 5: Summary of input and output factors for the flow conditions optimisation DSD

Experiment F14 was performed to validate the model and is not included in the DSD calculations. Experiments F15 and F16 are repeats of experiment 14 but with higher buffer concentration.

The conditions for F14, given in Table 5 drastically reduced the flow rate to 5 mI min ¹ and increased protein concentration to 10 mg ml ¹ . Protein volume was reduced to 4 ml to conserve material since it had a lower significance than protein concentration, which was prioritised. The pH was maintained at 7.1 since any lower was out of the buffering range for TRIS. The salt concentration, which was not a significant factor, was set at 50 mM. Under these conditions, a relatively high loading of 2.19 wt.% was obtained which roughly followed the prediction of the RSM model (Figure 11c). Immobilisation efficiency was still high at 39.38% but fell short of the model’s prediction of 59.6% (Figure 11d). A previous scoping experiment had predicted an even higher loading value with a higher buffer concentration, therefore experimental condition 14 was repeated but at higher buffer concentrations of 150 and 250 mM (experiments 15 and 16, respectively presented in Table 5). This showed a higher loading and immobilisation efficiency with increasing TRIS concentration as hypothesised, peaking at 150 mM (Figure 12).

As a result of the experiments above, the optimised immobilisation procedure was determined as follows:

After optimisation of the protein immobilisation procedure, as detailed above, the following changes were made to the above protocol: 1) the flow rate of CBM3A-mCherry was decreased from 100 to 5 pi min ¹ , 2) the CBM3A-mCherry volume was decreased from 5 to 4 ml, 3) the CBM3A-mCherry concentration was increased from 2 to 10 mg ml ¹ , 4) the buffer pH was decreased from 7.5 to 7.1, 5) the buffer KCI concentration was changed from 200 to 50 mM and 6) the buffer TRIS concentration was changed from 50 to 150 mM. To prioritise immobilisation yield over total loading, lower protein concentrations could be employed.

Example 4: Comparing performance of various CBM-fusion constructs under optimised conditions Having determined the optimal conditions for protein loading and immobilisation as shown in Examples 1 , 2 and 3, a number of different CBM-fluorescent protein constructs were loaded onto CSs under the optimised CS synthesis conditions and optimised flow conditions. This demonstrates the translatability of the process, i.e. that the optimisation undertaken for the CBM3-mCherry construct is applicable to other CBMs with other fluorescent proteins attached. Additionally, two control experiments were also conduced, 1) testing mCherry with no CBM attached to confirm immobilisation is due to the CBM and not any other effect (e.g. non-specific physical adsorption), and 2) mechanically ablating a CS to destroy the aligned porous structure.

Results and Discussion

The results for these experiments are presented in Figure 13. CBM3A-mCherry, CBM30-mOrange and CBM2a-mEGFP and the control experiments were all tested under the conditions optimised primarily to maximise loading (10 mg ml ¹ protein concentration), however mNeptune-CBM28 could only be concentrated to ~5 mg ml ¹ so was tested at that concentration instead. CBM2a-mEGFP was also tested at 5 mg ml ¹ to have a direct comparison with mNeptune-CBM28, as was mEGFP with no CBM as a further control. After flowing through the protein solutions, 10 ml of buffer (50 mM KCI, 150 mM TRIS, pH 7.1) was flushed through at a rate of 250 pi min ¹ , measuring the protein concentration in the eluent every 2 ml to assess the extent of protein leeching from the scaffolds. The results found that mCherry with no CBM had poor initial loading and immobilisation efficiency (40.9% and 2.02 wt.%, respectively) with very little retained after the 10 ml buffer flush (6.9% and 0.34 wt.%), compared with CBM3A-mCherry which had a much higher initial immobilisation efficiency and loading (79.4% and 5.24 wt.%, respectively) with far more retained after the 10 ml buffer flush (70.1% and 4.63 wt.%) than without the CBM (Figure 13a and 13b). This control was strong evidence that the immobilisation was indeed due to CBM-based binding and not a false positive due to another mechanism. The control experiment with a mechanically ablated CS also had a high initial immobilisation efficiency and loading at 91.3% and 5.94% respectively, however leeching was more pronounced with immobilisation efficiency and loading falling to 64.1% and 4.15 wt.% after the 10 ml buffer flush. The data also show that the CBMs fused with other fluorescent proteins also bind well to the CSs, demonstrating translatability of the process. In particular, CBM2a-mEGFP performed very well with an initial immobilisation efficiency of 91.8% and initial loading of 4.59 wt.%, falling to 78.7% and 3.94 wt.% respectively after the 10 ml buffer flush. By inference, other CBMs fused to other macromolecules (including enzymes) would also perform similarly well under these conditions.

Example 5: Demonstrating translatability to an enzyme and use in a continuous flow biocatalysis (CFB) setup

This experiment, applied the work of Examples 1 - 4, directly translates the process to an enzyme to demonstrate activity in a CFB setup. An w-transaminase (wTA) from B. megaterium (SC6394) was selected as a representative enzyme for this purpose due to its significant potential as an industrial biocatalyst. ooTA’s are pyridoxal phosphate (PLP) dependant enzymes that can transfer an amino group from a donor molecule to the carbonyl group of an acceptor molecule to produce a-chiral amines. Furthermore, the enzymatic activity of ooTA’s can be determined though a relatively simple photometric assay based on the detection of acetophenone production which absorbs strongly at 245 nm. The reaction scheme depicting the conversion of (S)-a-MBA into acetophenone via the CS immobilised CBM2a-ooTA enzyme is shown in Figure 14.

Method

A fusion construct consisting of CBM2a from C. cellulovorans (EngD) with the aforementioned wTA was designed and expressed - essentially switching out the mEGFP fluorescent protein for the wTA enzyme. CBM2a was employed as it displayed the best performance in Example 4. Employing the optimised CS synthesis and optimised in-flow immobilisation conditions (described above) with minor modifications, the CBM2a-ooTA constructs were immobilised to CSs. Loading and immobilisation efficiency was subsequently determined by measuring protein concentration before and after immobilisation via the Bradford method.

CBM2a-ooTA immobilisation conditions:

The CBM2a-ooTA construct was immobilised to optimised CSs following the optimised immobilisation procedure outlined above, with the following minor changes: 1) the CBM2a-ooTA volume was reduced to 3 ml and 2) the flow rate was reduced to 4 mI_ min ¹ . These changes were made to conserve limited material - allowing experiments to be done from a single production batch (eliminating batch-to-batch variability error). The concentration of the CBM2a-ooTA could be varied to prioritise either total loading or immobilisation efficiency. For instance, a protein concentration of ca. 10 mg ml ¹ resulted in a 3.99 wt.% loading and immobilisation efficiency of 62.1%. A lower protein concentration of 6.7 mg ml ¹ resulted in a lower loading of 2.34 mg ml ¹ but higher immobilisation efficiency of 81.4%.

Measuring loading and immobilisation efficiency using the Bradford method:

Loading and immobilisation efficiency was determined by measuring protein concentration before and after immobilisation via the Bradford method, employing BSA as a standard - before renormalizing by direct comparison to CBM2a-mEGFP which was run in parallel. Protein concentration was determined through the Bradford method using a standard BSA calibration curve, before being normalised based on the difference in measured concentration for the CBM2a-mEGFP construct via the Bradford method (1.7 mg ml ¹ ) and via its absorbance at 488 nm (4.98 mg ml ¹ ).

Measuring CBM2a-ooTA enzyme activity:

In order to demonstrate that CBM-tagged recombinant enzymes immobilised to CSs can be employed in a CFB set up, their activity in flow was measured and compared with the activity of the free enzyme in solution. The aforementioned photometric assay involving the conversion of (S)-a-MBA (donor) and pyruvate (acceptor) was employed due to its simplicity and robustness (Figure 14).

Determining activity of CBM2a-ooTA in solution:

In order to benchmark the enzymatic activity of the immobilised CBM2a-ooTA construct, its conversion efficiency in solution (i.e. , non-immobilised) was first determined. Briefly, to a 1.5 ml Eppendorf tube the following were added: 100 mI potassium phosphate (KPI) buffer solution (1M, pH 8), 400 mI H2O, 50 mI pyruvate (500 mM in H2O), 250 mI (S)-a- MBA (100 mM in H ₂ 0), 100 mI PLP (2 mM in H ₂ 0) and 100 mI of CBM2a-wTA (10 mg ml ¹ in 50 mM TRIS pH7.5, 200 mM KCI in H2O). The final concentrations were therefore: 0.1 M KPI (pH 8), 25 mM pyruvate, 25 mM (S)-a-MBA, 0.2 mM PLP, 20 mM KCI, 5 mM TRIS and 1 mg ml ¹ CBM2a-ooTA (14.9 mM). The mixture was briefly agitated and maintained at 30°C, before 4 mI of solution was removed at defined time points (typically 5 minute intervals), added to 196 mI of H2O (50x dilution) in a 96 well plate and absorbance at 245 nm measured. The concentration of acetophenone was then determined by referring to a calibration curve of the reactants (pyruvate, a-MBA) and products (acetophenone) at 245 nm. The measurements were taken in triplicate and a parallel control experiment with no enzyme was also conducted as a negative control.

Determining activity of CS-immobilised CBM2a-ooTA in flow:

To determine the activity of CS-immobilised CBM2a-ooTA in a CFB-type set up, the construct was first loaded onto optimised CSs under optimised flow conditions with minor modifications as shown above. Briefly, 3 ml of aqueous CBM2a-ooTA (10 mg ml ¹ , 149.1 mM) in 150 mM TRIS buffer (pH 7.5) and 50 mM KCI was loaded onto a CS by flushing through at a rate of 4 mI_ min ¹ at 4°C. The concentration before and after loading was determined via the Bradford method and normalised to correct for the difference in measured concentration of CBM2a-mEGFP via its absorbance at 488 nm and its concentration measured by the Bradford method. This normalisation was done to allow direct comparison of the loading behaviour of CBM2a-ooTA and the other CBM- FPs. After loading, a running buffer solution consisting of 0.1 M KPI (pH 8), 25 mM pyruvate, 25 mM (S)-a-MBA, 0.2 mM PLP was passed through the CBM2a-ooTA immobilised CS at 30°C and a rate of 200 mI min ¹ , with aliquots being sampled every 5 minutes (i.e., every 1 ml of flow through), diluted by a factor of 50 and absorbance measured at 245 nm. The acetophenone content was then determined by comparison to a calibration curve.

Results

The data showed similar initial loadings (2.34 vs. 1.94 wt. %) and immobilisation yields (81.4 vs. 95.5 %) for the CBM2a-ooTA and CBM2a-mEGFP constructs - demonstrating that the process is readily translatable without having to go through time-consuming optimisation for each new enzyme employed.

The activity of the free enzyme in solution was determined and found to stabilize at around 50 % conversion (50.71 % after 300 minutes), which was used as a basis for comparison with the performance of the CS immobilised CBM2a-ooTA in flow (Figure 15a).

Replicability and retention of immobilised protein

In three replicate experiments, CBM2a-ooTA was loaded onto the optimised CSs following the optimised procedure as detailed in Examples 2 and 3, respectively. A reactant mixture consisting of 25 mM pyruvate, 25 mM (S)-a-MBA, 0.2 mM PLP and 100 mM KPI buffer at pH 8 was then passed through the CSs at a rate of 200 pi min ¹ , and the production of acetophenone was monitored every 5 minutes (or every 1 ml) at 30°C (Figure 15b). The results show that after an initial lag period of about 40 minutes the conversion relative to free enzyme in solution approached 95 ±5 % with good reproducibility between the three replicate experiments. This confirms retention of activity of the enzyme when immobilised onto the internal channels of a wood-derived CS via CBM-based affinity binding.

Example 6: Demonstrating that a range of wood types can be employed

The experiment detailed in this example demonstrates that wood types other than Basswood may be employed to produce the CSs and that they function in an analogous manner. In order to demonstrate the principle Oak wood was investigated as it is a hardwood in contrast to Basswood which is a softwood.

Method

The optimised CS fabrication procedure (see Example 2 for details) was employed with Oak wood rather than Basswood to produce Oak-derived CSs. An aqueous solution of CBM3a-mCherry (3.62 mg ml ¹ ) was subsequently flushed through both the Oak- derived and Basswood-derived CSs in parallel, following the optimised procedure as detailed above. Impure CBM3a-mCherry obtained from a cell lysate was employed in this instance which adds further support to Example 7 below. By using impure CBM3a- mCherry obtained from a cell lysate it was not possible to use the optimal protein concentration of 10 mg ml ¹ as the process of concentrating the CBM3a-mCherry would have removed some impurities.

Results The Oak-derived CSs had similar appearance and characteristics to the Basswood- derived CSs (Figure 16a). The protein loading and initial immobilisation yield (based on two duplicate experiments for each type of wood) is presented in Table 6 below and shown in Figure 16b. optimal CS synthesis and protein immobilisation methods are employed.

The initial protein loadings for Basswood-derived and Oak-derived CSs were 1.95 and 1.60 wt. %, respectively. The initial immobilisation yields were 95.4% and 91.7%, falling to 92.0% and 87.5% after flushing with 5 ml of buffer, for Basswood-derived and Oak- derived CSs, respectively. This data demonstrates that Oak-derived CSs perform almost as well as Basswood-derived CSs. The finding that comparable levels of protein loading and immobilisation efficiency were achieved when using two types of wood that are so different indicates that the CS synthesis and protein immobilisation methods would work for other wood types. Based on this data it is reasonable to expect that many other kinds of softwoods and hardwoods could be employed using the methods set out in this specification.

Example 7: Demonstrating that CBM-fusion constructs can be directly immobilised to CSs from the cell lysate

After the recombinant production of an enzyme or protein from a host organism, costly and timely purification steps are typically employed in order to obtain a pure enzyme which may then be subject to immobilisation. Here, the inventors demonstrated that a CBM-fusion construct consisting of CBM3a and mCherry (see example 1 for details) was immobilised to an optimised CS scaffold (see example 2 for details) under optimised flow conditions (see example 3 for details) directly from the cell lysate without any other purification steps aside from brief centrifugation to remove insoluble cell debris. Method

Briefly, a CBM3a-mCherry fusion construct was expressed in E. coli before the cells were collected and subject to lysis via sonication. The lysed cells were briefly centrifuged to remove the majority of the insoluble cell debris, but other soluble impurities remained. This resulted in a solution with a concentration of about 3 mg ml ¹ . The solution was not subject to further concentration since such a step would also remove some soluble impurities. The solution was flowed through an optimised CS under optimised flow conditions (see example 3 for details), aside from the concentration being sub-optimal.

Results and Discussion

The protein loading (based on two duplicate experiments) was 1.7 wt.%, with an immobilisation yield of 97.6%, falling to 95.8% after a further 5 ml flush of buffer (Table 7). Higher loadings would likely be possible with higher initial protein concentrations, but would come at the expense of immobilisation yield. As a control, mCherry with no CBM attached was also flowed though in an analogous experiment, although a lower concentration of 0.5 mg ml ¹ was employed due to lower expression yield and since it was not possible to concentrate the sample without also removing impurities. This control experiment showed a much lower protein loading of 0.22 wt. % and lower immobilisation yield of 77.6 %, falling significantly to 61.9 % after a 5 ml flush of buffer indicating poor attachment. A visual inspection of the immobilised CSs also suggested that the CBM3a-mCherry CS was unsaturated and could accommodate further protein, whereas the mCherry control appeared saturated (Figure 17).

Table 7: Immobilisation yield and loading percentage obtained when non-purified cell lysate is used as the source of protein loading.

Example 8: Demonstrating that other enzyme immobilisation techniques can be employed and that non-aqueous solvents may be employed in a continuous flow biocatalysis (CFB) reaction. In this experiment, the inventors demonstrated that another enzyme (a lipase) can be immobilised to the CSs via a different mechanism (covalent coupling) and employed in a continuous flow reaction using a non-aqueous solvent (hexane) as the mobile phase.

Briefly, a number of CSs synthesised via the optimised synthesis procedure (see above) were functionalised with aldehyde groups via treatment with aqueous sodium periodate. A lipase enzyme was then covalently attached to the CSs via reaction between terminal amine groups on the enzyme and the aldehyde groups on the CSs, forming imine bonds. The imine bonds could then be selectively reduced to more robust amine bonds through treatment with aqueous sodium cyanoborhydride (Figure 18). All these steps were done in flow by flushing the solutions through the CSs at defined flow rates (see below for the method in greater detail).

A solution of 4-Nitrophenyl palmitate (NPP) in the organic solvent hexane could then be passed through the lipase-immobilised CSs, whereupon the lipase would enzymatically hydrolyse the NPP liberating 4-Nitrophenol. The production of 4-Nitrophenol could be monitored via UV-Vis spectrophotometry due to its intense yellow colouration (i.e. absorbance at 400 nm).

Method

A number of CSs synthesised via the optimised synthesis procedure (see example 2 above) were flushed with 20 ml of sodium acetate buffer (100 mM, pH 4) each at a rate of 200 pl_ min ^-1 to acidify the environment. Following this, the CSs were flushed with 20 ml of sodium periodate (10 mg ml ¹ ) in the same acetate buffer at a rate of 200 mI_ min ¹ . 20 ml of sodium phosphate buffer (50 mM, pH 7) was then flushed through the CSs to neutralise the environment. Following this, 5 ml of lipase B from Candida Rugosa (5 mg ml ¹ ) in the same phosphate buffer was flowed through the CSs at a rate of 4 mI_ min ¹ . Following this, the CSs were flushed with 5 ml of the same phosphate buffer to remove any non-attached lipase. The calculated immobilisation yield was 38.5 % with a loading of 0.26 wt. %, which could likely be improved through optimisation of the process. To improve attachment and reduce the extent of enzyme leeching, the CSs were then flushed with 5 ml of sodium cyanoborohydride (10 mg ml ¹ ) at 100 mI min ¹ to reduce the labile imine bonds with more stable amine bonds (Figure 18). The CSs were then flushed with 20 ml of the aforementioned sodium phosphate buffer (20 ml, 200 pi min ¹ ) to remove any excess sodium cyanoborohydride. The CSs were then flushed with 5 ml of hexane at a rate of 100 mI min ¹ to prime the scaffolds with the organic solvent. Following this, 5 ml of a 10 mM solution of 4-Nitrophenyl palmitate (NPP) in hexane was flushed through the CSs at a rate of 100 mI min ¹ to prime the CS with the precursor. NPP is a lipase substrate commonly used in assays as it produces a yellow colouration (absorbance at 400 nm) when hydrolysed by lipase. The production of yellow colouration may be easily monitored via UV-Vis spectrophotometry. Following this, a further 5 ml of 10 mM NPP in hexane was flown through the CSs at a rate of 5 mI min ¹ and collected. The NPP solutions flown through the lipase-immobilised CSs were then added to 0.1 M NaOH (1:9 ratio, organic to aqueous) and agitated to extract the yellow-coloured 4-nitrophenol into the aqueous phase. UV-Vis spectrophotometry was then performed on the aqueous phase and to determine the absorbance at 400 nm (Figure 19a). This data could then be used to calculate the percentage conversion of the NPP in the presence of the immobilised lipase.

Results and Discussion

As the solution of 4-Nitrophenyl palmitate (NPP) in hexane was passed through the CSs with lipase immobilised within, the lipase enzymatically hydrolysed the NPP thereby liberating 4-Nitrophenol. The production of 4-Nitrophenol was monitored via UV-Vis spectrophotometry (absorbance at 400 nm). With an NPP concentration of 10 mM and flow rate of 4 mI min ¹ , equating to a residence time of approximately 4 hours, the percentage conversion was 3.28%. A control experiment with no immobilised lipase enzyme showed a much lower conversion of 0.06% as can be seen by comparison of the UV-Vis absorbance profiles (Figure 19a). Visual inspection of the CSs also found that the lipase-immobilised scaffold turned yellow due to exposure to 4-Nitrophenol, whereas the lipase-free control had not (Figure 19b). For comparison, the activity of free lipase in a 10mM solution of NPP in hexane was determined (Figure 19c and 19d). After 4 hours, the conversion was 5.17% - suggesting that the immobilised enzyme had about 63.5 % of the activity of the free enzyme in solution. It should be noted that this process may be optimised to improve the reaction dynamics and would likely improve the performance of the immobilised enzyme significantly. Parameters that would likely have a significant effect on the efficacy of the immobilised enzyme and would therefore be good candidates for optimisation include: flow rate (lower flow rate likely more optimal due to increased residence time), temperature (immobilised enzymes can tolerate high temperature and perform better than enzymes in solution) and the conditions employed to introduce aldehyde groups onto the cellulose (e.g, sodium periodate concentration and volume) which would increase enzyme loading. It will be appreciated that numerous modifications to the above-described method may be made without departing from the scope of the invention as defined in the appended claims. For example, although in the examples a w-transaminase and a lipase are immobilised on the macromolecule scaffolds of the invention, the skilled person will appreciate that any other protein, for example enzyme, may suitably be immobilised on the scaffolds of the invention. Furthermore, although immobilisation of the exemplified proteins is shown using CBMs or covalent amine bonds, the skilled person will appreciate that other immobilisation techniques may be used. For instance, trimethoxy silanes (TMS) readily react with cellulose and can introduce various functional groups to the exposed cellulose surfaces. These include epoxy-TMS, TMS-methacrylate and mercaptopropyl-TMS which would introduce epoxide, methacrylate and thiol functional groups on the cellulose surface for covalent immobilisation with proteins or other macromolecules.

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