Direct functionalisation of materials

Abstract

The present disclosure relates to the generation of functionalised matrices comprising self- aggregating protein particles absorbed, combined or bound to the porous matrix. More particularly, the disclosure relates to matrices comprising p40 containing protein nanoparticles which modified matrices exhibit functional activity.

Description

[0001] Direct Functionalisation of Materials

[0002] All documents cited or referenced herein, and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference in their entirety.

[0003] The present application claims priority from Australian Provisional Patent Application No 2024902318 filed on 25 July 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] The entire content of the electronic submission of the sequence listing is incorporated by reference in its entirety for all purposes.

[0005] Field of the disclosure

[0006] The present disclosure relates to the generation of functionalised matrices comprising self-aggregating protein particles absorbed, combined or bound to the porous matrix. More particularly, the disclosure relates to matrices comprising p40 containing protein nanoparticles which exhibition functional activity.

[0007] Background

[0008] Biofunctionalisation is a pivotal technique in biomaterial design. It involves modifying material surfaces to incorporate functional or reactive groups. These surfaces incorporate functional moieties conferring specific functionalities to the material without altering their key physical properties. Biofunctionalisation bridges the gap between biomaterials and biomolecules, enabling safer and more effective biomedical and biotechnological applications. Based on the nature of interaction between the material and biomolecule, the mode of biofunctionalisation can be non-covalent (physical adsorption, entrapment), covalent (comprising chemical crosslinker) or a combination of both. Selection of the right biofunctionalisation strategy is highly essential for the maintenance of the biomolecule’s activity and reusability of functionalised material.

[0009] Both the covalent and non-covalent mode of functionalisation have their own advantages and limitations. Non-covalent functionalisation techniques including entrapment, encapsulation and physical adsorption like TT-TT stacking, electrostatic interaction are gentle and uncomplicated methods that only slightly harms the biomolecules and the biomaterial substrate. However, non- covalent interactions may result in less stable bonds between the biomolecules and the material surface. Overtime, these interactions can weaken, affecting the longevity of the biofunctionalised material. Furthermore, non-covalent modifications are frequently reversible. This flexibility makes modification simple, but in some cases, it can also cause desorption of biomolecules, impacting the material’s performance. Zhang et al., (2010) Langmuir, 26(9), 6083-6085; Zhang et al., (2010) Small, 8(1), 154-159 demonstrated the necessity of surface modification of graphene oxide (GO) to facilitate stable functionalisation of enzymes. In their previous work, GO was functionalised with horseradish peroxidase (HRP) through a weak electrostatic interaction between GO surface and HRP. However, the enzyme’s activity was negatively impacted due to electrostatic interaction even though the enzyme loading on GO was higher than classic material. Moreover, the GO-immobilised enzyme's stability was insufficient for practical application. An additional surface modification of GO to chemical reduced graphene oxide (CRGO) have been reported to enhance the enzyme adsorption to a tenfold higher than GO. Also, enzyme functionalised on CRGO exhibited higher stability, and relative activity compared to GO-enzyme conjugate.

[0010] Therefore, the binding efficiency, reversibility and stability of biomolecules are the challenges to be addressed for an efficient non-covalent mode of biofunctionalisation. Unlike non-covalent methods, covalent methods ensure stability and higher binding affinity mediated by crosslinking agents which promotes spatial distribution of biomolecules by reacting with the substrate’s surface and enhancing the longevity of the attached biomolecules. Different approaches have been employed forthe covalent functionalisation of various materials for myriad of applications including food packaging, water treatment, textile and biomedical field.

[0011] However, the use of harsh chemical crosslinkers, which can introduce toxicity, and the need for chemical activation of materials lacking reactive groups, make the covalent biofunctionalisation process complex. For instance, an extensive review by Balamurali et al., (2022) Molecules, 27(23), 8124) summarised the potential limitations of utilising natural or synthetic crosslinkers for protein functionalisation, highlighting their tendency to damage the protein structure and the necessity to remove residual cross-linking agents.

[0012] Another growing avenue of carrier-free enzyme immobilisation is in vivo produced self- immobilisates through catalytically active inclusion bodies (CatIBs). Overexpression of heterologous gene results in the formation of self-assembled insoluble protein particles called inclusion bodies (IBs). Despite the general view that inclusion bodies (IBs) are undesired inactive substances, many findings have overlooked this fact. Various strategies have been reported to produce CatIBs, particularly through the utilisation of diverse aggregation-inducing tags and alterations in production parameters.

[0013] There is a need in the art for methods that overcome the challenges associated with the foregoing biofunctionalisation methods and which provides for cross linker free surface functionalisation of materials that confer functionality without altering their physical properties. Summary of the Disclosure

[0014] The present disclosure is based on exploiting the properties of protein particles within inclusion bodies (IB) typically formed during overexpression of recombinant genes in bacteria to biofunctionalise various matrices.

[0015] As herein described, the solubilisation-reaggregation property of protein nanoparticles (PNPs) was successfully used for biofunctionalisation of various matrices including sheets and particles (e.g. beads or rods or the like). The PNP preferably comprises an aggregating part comprising a p40 protein which may be linked to a functional part. The functional part which may have various activities including enzymatic activity, detectable activity, antibody activity, hydrolyse activity, carbohydrate binding activity, phosphorylase activity, isomerase activity, metal binding activity or antibacterial activity. In particular, the solubilisation-reaggregation property of p40 IBs was utilised for biofunctionalisation of various matrices as exemplified using fluorescent p40 IBs (e.g. mCherryP4o and eGFPpw) and enzyme p40 IBs (e.g. BactXylBP4o and BacAmyP4o), thus demonstrating the flexibility and potential of this strategy for various applications.

[0016] The solubilisation-reaggregation strategy provides a versatile platform technology for the production of biologically active PNPs with diverse functionalities. Biofunctionalisation of materials based on the "solubilisation-reaggregation" property of p40 offers a more unified, simplified, and potentially universal strategy for the direct immobilisation of industrially important enzymes (in the form of aggregating particles) onto solid materials (e.g. magnetic, silica and glass beads), thereby enabling enzyme recycling and improving stability. Moreover, this approach provides for direct functionalisation of materials not only with enzymes but also with bioactive peptides (e.g., antimicrobial, anticancer, inflammatory, antioxidant, antiviral, etc.) and other biomolecules.

[0017] In a first aspect, there is provided a functionalised matrix comprising:

[0018] (i) a protein nanoparticle (PNP) having an aggregating part capable of forming or aggregating into a protein particle and a functional part providing functional activity to the matrix; and

[0019] (ii) a porous matrix; wherein the PNP is absorbed, combined, bound to, or contained within the porous matrix.

[0020] In one example, the PNP is absorbed, combined, bound to, or contained within the porous matrix following solubilisation of the PNP and reaggregation in the presence of the matrix.

[0021] In one example, the PNP is an insoluble protein aggregate.

[0022] In one example, absorption, combining and containing the PNP within the porous matrix is achieved by causing the PNP to be solubilised with a solubilising agent that permits solubilisation of the PNP without causing denaturation of the PNP. The inventors tested a number of solubilising agents and unexpectedly found that solubilisation of the PNP without resulting denaturation was only successfully achieved using guanidinium chloride (GdmCI). In one example, the solubilising agent is guanidinium chloride (GdmCI). In a particular example, the solubilising agent is 3M GdmCI. In some examples, the solubilising agent further comprises sodium dodecyl sulfate (SDS) or N-lauroylsarcosine sodium salt (NLS).

[0023] In certain examples, the PNP is solubilised for a period of between 30 mins and 3 hours, or between 60 mins and 2 hours. In one example, the PNP is solubilised for a period of about 2 hours.

[0024] In a further example, reaggregation occurs when combined with the matrix.

[0025] In some examples, the aggregating part of the PNP is a synthetic peptide or a naturally occurring peptide. The aggregating part may be a known protein or part thereof or an artificially formed protein or peptide that when expressed in a cell forms aggregates. In one example, the aggregating part of the PNP is selected from an Auxiliary Activity Family 10 (AA10) enzyme (formerly CBM33), http : / / ww . crazy . o rg / AA In one example, the aggregating part is an oxidative enzyme of the AA10 family. In one example, the aggregating part is a protein comprising or consisting of a sequence or functional part thereof set forth in Table 1 . In one particular example, the aggregating part is a protein comprising or consisting of a sequence or functional part thereof selected from a sequence set forth in SEQ ID NOs: 1-4 or 27-124. In a further particular example, the aggregating part is a p40 protein or peptide or analogue thereof.

[0026] In one example, the aggregating part comprises or consists of a p40 sequence set forth in: VFPATRTYACYVDGKVHGNGGDLNMINPACLDALAISGNYQFWNWFGNLISNAGGRHREIIPD GKLCGPTASFDGMNQARTDWWTTRLQPGATITVRVNAWAPHPGTWYLYVTRDGWDPTQPL KWSDLEPTPFSQVTNPPINSSGPDGAEYSWQVQLPNKQGRHIIYMIWQRSDSPEAFYNCSDV YFGSGPIAYEFGDPREGG (SEQ ID NO:1) or a sequence at least 60% identical thereto.

[0027] In one example, the p40 protein comprises or consists of a sequence at least 65% identical to the sequence of SEQ ID NO:1. In another example, the p40 protein comprises a sequence at least 65%, at least 67%, at least 70%, at least 72%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 98% or at least 99% identical to the sequence of SEQ ID NO:1.

[0028] In one example, the p40 protein comprises or consists of a sequence set forth in SEQ ID NO:1.

[0029] In one example, the p40 protein comprises or consists of the sequence set forth in: VSPATRTYACYVDGRANGGGDLNPTNPACVAAVAQGGKQPLWDFFAVLQSNAGGNHRAIIP DGQLCGGGTTKYAAYNAARTDWPTTQLQSGGTMQFRYNAWAPHPGTWYQYITRDGYDPTQ PLKWSDLEATPFDQVTNPPTQGGPSGSEYYWNTRLPVKQGRHIIYSIWQRSDSPEAFYNCVD VQFGG (SEQ ID NO:2). In one example, the p40 protein comprises or consists of the sequence set forth in: TFPSTRTHACYVDGKAGGGGDLNPQNPACKAAVAIGGKQPLWDWFGNLISNAGGRHREIIPD GKLCGPTAKYDGYNLARTDWPTTQLQSGAAITFRYNAWAPHPGTWSQYITRDGWNPNQPLK WSDLEATPFNSVTNPPINGSGPEGAEYTWPGVLPRKTGRHIIYSIWQRSDSPEAFYNCSDVNF GGS (SEQ ID NO:3).

[0030] In one example, the p40 protein comprises or consists of the sequence set forth in: TYPATRTYACYVDGKAGGQGGDLHPTNPACVAAVAEGGKNPLWNWFGNLISNAAGRHREIIP DGKLCGPTALFDAYNMAHDEWPTTSLQAGSSITIRYNAWAPHPGTWYQYVTKDGWDPSQPL KWSDLEPVPFDTVTNPPINGTGPEGPEYTWTAQLPANKSGRHIIYSIWQRSDSPEAFYNCSDV VFDGG (SEQ ID NO:4).

[0031] In a further example, the p40 sequence comprises or consists of a p40 fragment. In one example, the part is a functional fragment of the sequence of SEQ ID NO:1.

[0032] In one example, the p40 fragment comprises or consists of the sequence: VFPATRTYACYVDGKVHGNGGDLNMINPACLDALAISGNYQFWNWFGNLISNAGGRHREIIPD GKLCGPTASFDGMNQARTDWWTTRLQPGATITVRVNAWAPHPGTWYLYVTRDGWDPTQPL KWSDLEPTPFSQVTNPPINSSGPDGAEYSWQVQLPNKQGRHIIYMIWQRSDSPEAFY (SEQ ID NO:21).

[0033] In one example, the p40 fragment comprises or consists of the sequence: VFPATRTYACYVDGKVHGNGGDLNMINPACLDALAISGNYQFWNWFGNLISNAGGRHREIIPD GKLCGPTASFDGMNQARTDWWTTRLQPGATITVRVNAWAPHPGTWYLYVTRDGWDPTQPL KWSDLEPTPFSQVTNPPINSSGPDGAEYSWQVQLPNKQGR (SEQ ID NO:22).

[0034] In one example, the p40 fragment comprises or consists of the sequence: VFPATRTYACYVDGKVHGNGGDLNMINPACLDALAISGNYQFWNWFGNLISNAGGRHREIIPD GKLCGPTASFDGMNQARTDWWTTRLQPGATITVRVNAWAPHP (SEQ ID NO:23).

[0035] In one example, the p40 fragment comprises or consists of the sequence: VFPATRTYACYVDGKVHGNGGDLNMINPACLDALAISGNYQFWNWFGNLISNAGGRHREIIPD GKLCGPTASFDGMNQARTDWWTTRLQP (SEQ ID NO:24).

[0036] In one example, the p40 fragment comprises or consists of the sequence: VFPATRTYACYVDGKVHGNGGDLNMINPACLDALAISGNYQFWNWFGNLI (SEQ ID NO:25).

[0037] In one example, the p40 fragment comprises or consists of the sequence: VFPATRTYACYVDGKVHGNGGDLNMINP (SEQ ID NO:26).

[0038] In a further example, the sequence according to any one of SEQ ID NOs:21 to 26 is fused or linked to a functional part as described herein. In a further example, the sequence according to any one of SEQ ID NOs:21 to 26 is fused or linked to mCherry, alpha-amylase (BactAmy), alpha glucan phosphorylase (aGP) or phosphoglycolate phosphatase (PGP). In one example, the functional part of the PNP is selected from mCherry, enhanced green fluorescence protein (eGFP), alpha-amylase (BactAmy), alpha glucan phosphorylase (aGP) or phosphoglycolate phosphatase (PGP), phosphoglucomutase (PGM), glucose-6-phosphate isomerase (PGI), beta-galactosidase ( -Gal), frutalin (FTL), microvirin (MVN), metallothionein (metallo), anti-His6 nanobody (Anti-His), ZXR-2, (a lytic peptide derived from the N-terminal sequence of mauriporin, a scorpion venom toxin Zhou X-R et al., (2016) Biochemica et Biophysica Acta (BBA) - Biomembranes vol 1858(8): 1914-1925)), BactXylB (Xylanase from Bacillus subtilis) F6PE, fructose 6-phosphate 4-epimerase (F6PE) and Tagatose 4-epimerase (T4E).

[0039] In one example, the functional part of the PNP may comprise an enzyme, peptide, a lectin, an antigen-binding molecule, a metallothionein, a fluorescent label or other biomolecule. In one example, the functional part of the PNP may comprise a red or green fluorescence, a hydrolase, a phosphorylase, a glycosidase, or an isomerase.

[0040] In one example, the functional part is capable of binding to or being bound by a target compound.

[0041] In a further example, the aggregating part and the functional part are fused together. In a further example, the aggregating part and the functional part are joined by a linker. In one example, the linker is a GS linker. In another example, the linker is a G-G linker. In a particular example, the linker comprises or consists of the sequence (GGGGS)3 (SEQ ID NO:125).

[0042] In one example, the functional part is N-terminal to the aggregating part of the PNP.

[0043] In one example, the PNP is capable of being solubilised without denaturing.

[0044] In one example, the PNP is recombinantly produced. In one example, the PNP is synthetic.

[0045] In another example, the PNP comprises one or more different functional parts capable of binding one or more different target components

[0046] The target compound may be a compound on which the functional part has activity (e.g. a substrate for an enzyme).

[0047] In another example, the functionalised matrix comprises two or more different p40 proteins described herein. In another example, the p40 proteins are contiguous or separated by a linker.

[0048] In one example, the PNP comprises the sequence of SEQ ID NO:5. In one example, the PNP comprises the sequence of SEQ ID NO:6. In one example, the PNP comprises the sequence of SEQ ID NO:7. In one example, the PNP comprises the sequence of SEQ ID NO:8. In one example, the PNP comprises the sequence of SEQ ID NO:9. In one example, the PNP comprises the sequence of SEQ ID NO:10. In one example, the PNP comprises the sequence of SEQ ID NO:11. In one example, the PNP comprises the sequence of SEQ ID NO:12.. In one example, the PNP comprises the sequence of SEQ ID NO:13. In one example, the PNP comprises the sequence of SEQ ID NO:14. In one example, the PNP comprises the sequence of SEQ ID NO:15. In one example, the PNP comprises the sequence of SEQ ID NO:16. In one example, the PNP comprises the sequence of SEQ ID NO:17. In one example, the PNP comprises the sequence of SEQ ID NO:18. In one example, the PNP comprises the sequence of SEQ ID NO:19. In one example, the PNP comprises the sequence of SEQ ID NO:20.

[0049] In another example, the functionalised matrix is recyclable or reusable.

[0050] In a second aspect, there is provided a method of forming a functionalised matrix, the method comprising:

[0051] (i) producing a protein nanoparticle (PNP) having an aggregating part capable of forming or aggregating into a protein particle and a functional part providing functional activity to the matrix; and’

[0052] (ii) combining the PNP with a matrix wherein the PNP is in a solubilised form and reaggregation occurs in the presence of the matrix such that the PNP is absorbed, combined, bound to, or contained within the matrix.

[0053] In a third aspect, there is provided a method of forming a functionalised matrix, the method comprising:

[0054] (i) harvesting insoluble protein aggregates, the aggregates comprising a protein nanoparticle (PNP), the PNP having an aggregating part capable of forming or aggregating into a protein particle and a functional part providing functional activity to the matrix;

[0055] (ii) solubilising the insoluble protein aggregates to yield the PNP;

[0056] (iii) combining the solubilised PNP with a matrix; and

[0057] (iv) allowing the solubilised PNP to reaggregate in the presence of the matrix for a time sufficient to allow the PNP to be absorbed, combined, bound to or contained within the matrix.

[0058] In one example, the aggregating part of the PNP is described herein. In a further example, the aggregating part is a p40 protein described herein.

[0059] In one example, the functional part is capable of binding to or being bound by a target compound.

[0060] In one example, the PNP is capable of being solubilised without denaturing. In one example, the method comprises solubilising the insoluble protein aggregates with a solubilising agent. In a particular example, the solubilising agent is an agent that permits solubilisation and reaggregation of the PNP. In one example, the solubilising agent is guanidinium chloride (GdmCI). In some examples, the solubilising agent further comprises sodium dodecyl sulfate (SDS) or N-lauroylsarcosine sodium salt (NLS). In certain examples, the PNP is solubilised for a period of between 30 mins and 3 hours, or between 60 mins and 2 hours. In one example, the PNP is solubilised for a period of about 2 hours.

[0061] In one example, the matrix is a porous matrix.

[0062] In one example, the method further comprises providing a nucleic acid molecule encoding a PNP of the disclosure to a cell and allowing the cell to express the nucleic acid molecule to form an insoluble protein aggregate and recovering the insoluble protein aggregate.

[0063] In one example, the PNP is cloned into a vectorwhich is expressible in a cell. In a further example, the nucleic acid is provided in a suitable construct, for example a vector, plasmid, virus or any other suitable expression means.

[0064] In one example, the cell is a bacterial cells or a yeast cell. In one example, the plasmid is pDuet:NP40NLCPA. The sequence of this plasmid is described in US 2012 / 0009624, the entire contents of which are incorporated by reference herein.

[0065] In one example, the functional part of the PNP is derived from a bacterium. In one example, the bacterium is selected from the group consisting of Anaplasma marginale, Aequorea Victoria, Bacillus licheniformis, Bacillus subtilis, Dictyoglomus turgidum, Thermus Thermophilus, Archaeoglobus profundus, Artocarpus incisa, Microcystis aeruginosa, Pisum sativum, Sulfolobus acidocaldarius and Androctonus mauritanicus.

[0066] In one example, the functional part of the PNP is selected from mCherry, enhanced green fluorescence protein (eGFP), alpha-amylase (BactAmy), alpha glucan phosphorylase (aGP) or phosphoglycolate phosphatase (PGP), phosphoglucomutase (PGM), glucose-6-phosphate isomerase (PGI), beta-galactosidase ( -Gal), frutalin (FTL), microvirin (MVN), metallothionein (metallo), anti-His6 nanobody (Anti-His), ZXR-2 , BactXylB, F6PE, fructose 6-phosphate 4- epimerase (F6PE) and Tagatose 4-epimerase (T4E).

[0067] The functional part of the PNP may comprise protein A, protein G, protein L, an antigen binding molecule (e.g. antibody or aptamer), a single chain antibody, avidin, streptavidin, an enzyme, an inhibitor, an antigenic determinant, an epitope, a binding site, a lectin, a cellulose binding protein, a polyhistidine, an oligohistidine, a receptor, a hormone, a signalling molecule, a polypeptide with specific or group specific binding capabilities, or a combination thereof.

[0068] The functional part of the PNP may comprise an enzyme, peptide or a fluorescent label.

[0069] A suitable matrix according to the disclosure may include any matrix material which is capable of being functionalised, in particular a material to which a protein nanoparticle of the disclosure can be absorbed, combined or bound to. In one example, the matrix is a porous or semi-porous material. In a further example, the matrix material is a natural or synthetic zeolite, silica, glass wool, metal-based fibre, a pad, or porous bead. In one example, the matrix material is selected from a polypropylene, a cellulose, a cupro / polyethylene terephthalate (PET), PET microfibre, PET, a polyethylene, a methacrylate, a dextran or polystyrene.

[0070] In particular examples, the matrix is selected from a non-woven PP fibre, Microline™ - CBSP060, Microline™ - CBSP100, Microline™ - CBSP097, Microline™ - PMAP080, Microline™ - PMAP090, Microline™ - PSCP250, BioCradle, Cytodexl , PE frit, Immobeads 150P or Bio-Beads.

[0071] In one example, reaggregation of the solubilised PNP occurs in the presence of the matrix. In one example, the method comprises reaggregating the solubilised PNP in the presence of the matrix with water.

[0072] In one example, the solubilisation and reaggregation of the PNP and matrix comprises agitation. In one example, the solubilised p40 nanoparticles and matrix are combined for at least 5 min. In one example, the reaggregation process is for at least 5 min.

[0073] In a fourth aspect, there is provided a method for separating at least one target component or compound from a sample or mixture comprising the target component or compound, the method comprising:

[0074] (i) combining the functionalised matrix comprising the PNP according to the first aspect or prepared according to the second or third aspect, with a sample or mixture containing the target component or compound; and

[0075] (ii) allowing a target component in the sample or mixture to bind to the functional part of the PNP.

[0076] In a fifth aspect, there is provided a method for enriching or separating at least one target component or compound from a sample or mixture comprising the target component or compound, the method comprising combining the functionalised matrix comprising the PNP according to the first aspect with a sample or mixture containing the target component or compound whereby enriching the at least one target component or compound from the sample or mixture.

[0077] In one example, the functionalised matrix is prepared according to the second or third aspect described herein.

[0078] Preferably, the method further comprises recovering the target component from the functionalised matrix once bound. The method according to the present disclosure can be used to enrich at least one desired component within a mixture by separating at least one undesired target component from the mixture or sample.

[0079] Preferably, when a mixture is contacted with the functionalised matrix the target component selectively binds, or is selectively bound by, the functional part of the PNP. The mixture may comprise any suspension, dispersion, solution or combination thereof of any biological extracts or derivatives thereof. For example, the mixture may include blood, blood plasma, blood serum, blood derived precipitates or supernatants, animal extracts or secretions, milk, colostrum, whey or any other milk derived product or fraction thereof, fermentation broths, liquids or fractions thereof, cell lysates, cell culture supernatants, cell extracts, cell suspensions, viral cultures or lysates, plant extracts or fractions thereof.

[0080] The target component may comprise a protein, a peptide, a polypeptide, an immunoglobulin, biotin, an inhibitor, a co-factor, a substrate, an enzyme, a receptor, a monosaccharide, an oligosaccharide, a polysaccharide, a glycoprotein, a lipid, a nucleic acid, a cell or fragment thereof, a cell extract, an organelle, a virus, a biological extract, a hormone, a serum protein, a milk protein, a milk-derived product, blood, serum, plasma, a fermentation product a macromolecule or any other molecule or any combination or fraction thereof. The biological extract may be derived from any plant, animal, microorganism or protista.

[0081] The target component may be a desired target component or an undesired target component. The undesired target component may be a contaminant.

[0082] The target component may be recovered from the functionalised matrix to which the target component is bound. The recovery of the target component may be via at least one elution step wherein the binding of the target component to the protein is weakened, disrupted, broken or competitively substituted.

[0083] The desired or the undesired component may comprise a protein, a peptide, a polypeptide, an immunoglobulin, biotin, an inhibitor, a co-factor, a substrate, an enzyme, a receptor, a monosaccharide, an oligosaccharide, a polysaccharide, a glycoprotein, a lipid, a nucleic acid, a cell or fragment thereof, a cell extract, an organelle, a virus, a biological extract, a hormone, a serum protein, a milk protein, a milk-derived product, blood, serum, plasma, a fermentation product a macromolecule or any other molecule or any combination or fraction thereof. The biological extract may be derived from any plant, animal, microorganism or protista.

[0084] In some examples, the functionalisation efficiency of the functionalised matrix is greater than or equal to 85%, greater than or equal to 87%, greater than or equal to 90%, greater than or equal to 92%, greater than or equal to 95%, greater than or equal to 97%, greater than or equal to 99% or 100%.

[0085] In some examples, the enzyme activity of the functionalised matrix is greaterthan 100%, greater than 110%, greater than 120%, greater than 130%, greater than 140%, greater than 150%, greaterthan 160% or greaterthan 170% compared to its activity priorto functionalisation (e.g. free p40). In some examples, the activity of the enzyme is maintained over repeated recycling.

[0086] In a sixth aspect, there is provided the functionalised matrix according to the first aspect for use or when used in a method of medical treatment. In one example, the method of medical treatment is wound healing. In one example the method of medical treatment is tissue regeneration.

[0087] In a seventh aspect, there is provided a kit for affinity separation comprising the functionalised matrix according to the first aspect and instructions for carrying out affinity separation. In one example, the kit comprises instructions for use in an in vitro diagnostic applications, tissue regeneration (e.g. such as treatment of burns), wound healing and organoid culture.

[0088] In order that the present invention may be more clearly understood, preferred embodiments will be described with reference to the following drawings and examples.

[0089] Description of the Figures

[0090] Figure 1 shows (A) SDS-PAGE gel profile of different p40_IBs after purification. The samples were diluted 1 :1 with 2X Laemmli sample buffer and 10 pL of each sample was loaded onto the gel and stained with SimplyBlue™ SafeStain. Lane M, molecular weight marker; 1 , p40 (~24.4 kDa); 2, BactXylBP4o (~47 kDa); 3, mCherryP4o (50.4 kDa); 4, eGFPP4o (50.8 kDa); 5, PGiP4o (~70 kDa); 6, PGMP4o (72 kDa); 7, BactAmyP4o (~82.4 kDa); 8, aGPP4o (~89.2 kDa), (B) Target protein content estimation of different p40 based IBs.

[0091] Figure 2 shows CLSM images of bare and functionalised synthetic PP fibre (different regions). Phase contrast image in the left panel; overlay image in the middle and right panel is fluorescence channel. (A) PP fibre treated with soluble eGFP (which fluoresces green) and (B) eGFPP4o- functionalised PP fibre. (C) PP fibre treated with soluble mCherry (which fluoresces red) and (D) mCherryP4o-functionalised PP fibre.

[0092] Figure 3 shows FESEM images of functionalised PP fibre at different magnifications. (A) Bare PP fibre treated with soluble mCherry. (B) Functionalised PP fibre containing aggregated mCherryP4o (circled).

[0093] Figure 4 shows CLSM images of bare and functionalised CBSP060 cellulose sample pad (different regions). Phase contrast image in the left panel; overlay image in the middle and right panel is fluorescence channel. (A) PP fibre treated with soluble mCherry and (B) CBSP060 after functionalisation with mCherryP4o.

[0094] Figure 5 shows CLSM images of bare and functionalised CBSP100 cellulose sample pad (different regions). Phase contrast image in the left panel; overlay image in the middle and right panel is fluorescence channel. (A) CBSP100 fibre treated with soluble mCherry and (B) CBSP100 after functionalisation with mCherryP4o. The fibre filaments bearing the fluorescent aggregates indicating the successful functionalisation.

[0095] Figure 6 shows FESEM images of CBSP060 fabric. (A) Bare CBSP060 treated with soluble mCherry at different magnifications. (B) Surface texture of functionalised CBSP060 bearing aggregated mCherryP4o (circled).

[0096] Figure 7 shows FESEM images CBSP100 fabric. (A) Bare CBSP100 treated with soluble mCherry at different magnifications. (B) Surface texture of CBSP100 after functionalisation bearing aggregated mCherryP4o (circled).

[0097] Figure 8 shows CLSM images of bare and functionalised CBSP097 cellulose / PET multilayer fabric (different regions). Phase contrast image in the left panel; overlay image in the middle and right panel is fluorescence channel. (A) CBSP097 treated with soluble mCherry and (B) mCherryP4o functionalised CBSP097 showing that fibre filaments bearing the fluorescent aggregates indicating the successful functionalisation.

[0098] Figure 9 shows FESEM images of CBSP097 cellulose / PET multilayer fabric. (A) Bare CBSP097 treated with soluble mCherry at different magnifications. (B) Images showing the tightly bound aggregates (circled) with the fibre filaments after mCherryP4o functionalisation.

[0099] Figure 10 shows CLSM images of bare and functionalised PMAP080, PET absorbent pad (different regions). Phase contrast image in the left panel; overlay image in the middle and image in the right panel shows fluorescence channel. (A) PMAP080 treated with soluble mCherry and (B) mCherryP4o-functionalised PMAP080 showing the fluorescent (red) filaments containing the aggregates.

[0100] Figure 11 shows FESEM images of PMAP080 matrix. (A) PMAP080 PET microfibers treated with soluble mCherry at different magnifications. (B) Images showing the adhering aggregates (circled) of mCherryP4o over the fibre filaments after functionalisation.

[0101] Figure 12 shows CLSM images of bare and functionalised PMAP090, PET absorbent pad (different regions). Phase contrast image in the left panel; overlay image in the middle and image in the right panel shows fluorescence channel. (A) PMAP090 treated with soluble mCherry and (B) mCherryP4o-functionalised PMAP090 showing the fluorescent (red) filaments containing the aggregates. Figure 13 shows FESEM images of PMAP090 PET fabric. (A) Fibertreated with soluble mCherry at different magnifications, images showing intact structure and smooth surface of bare fibres. (B) Images showing cross-linked fibres bound together by mCherryP4o aggregates (circled) after functionalisation.

[0102] Figure 14 shows CLSM images of PSCP250 conjugate pads (different regions). Phase contrast image in the left panel; overlay image in the middle and image in the right panel shows fluorescence channel. (A) PSCP250 treated with soluble mCherry and (B) Conjugate pad after functionalisation with mCherryP4o showing the filaments carrying distinct fluorescent aggregates.

[0103] Figure 15 shows FESEM images of PSCP250 conjugate pad. (A) material treated with soluble mCherry at different magnifications, images showing intact structure and smooth surface of filaments. (B) images showing bulged, irregular texture of PSCP250 filaments covered with mCherryP4o aggregates (circled) after functionalisation.

[0104] Figure 16 shows CLSM images of macroporous cellulosic BioCradle beads. Phase contrast image in the left panel; overlay image in the middle and image in the right panel shows fluorescence channel. (A) Images showing beads treated with soluble mCherry. The absence of fluorescence in right panel images indicate the inability of bare BioCradle beads to take up soluble mCherry and (B) BioCradle beads after functionalisation with mCherryP4o showing the distinct fluorescent (red) aggregates from surface to inner core.

[0105] Figure 17 shows FESEM images of macroporous BioCradle beads. (A) beads treated with soluble mCherry at different magnifications, images showing sharp structure of beads with intact pores. (B) images showing functionalised beads bearing mCherryP4o aggregates stuck into their pores (circled).

[0106] Figure 18 shows CLSM images of microporous dextran beads, Cytodexl . Phase contrast image in the left panel; overlay image in the middle and image in the right panel shows fluorescence channel. (A) images showing transparent Cytodexl beads treated with soluble mCherry which showed negligible fluorescence and (B) Cytodex beads after functionalisation with mCherryP4o showing the distinct fluorescent aggregates around the surface of the beads. Figure 19 shows FESEM images of Cytodexl . (A) beads treated with soluble mCherry. Treated beads retained their original smooth surface. (B) images showing functionalised beads bearing mCherryP4o aggregates embedded on their surface (circled).

[0107] Figure 20 shows CLSM images of sintered porous PE frits. Phase contrast image in the left panel; overlay image in the middle and image in the right panel shows fluorescence channel. (A) images showing PE frits treated with soluble mCherry displaying no fluorescence. (B) after functionalisation with mCherryP4o, the frits showed the presence of fluorescent aggregates within their sintered structures.

[0108] Figure 21 shows CLSM images of Immobeads 150P. Phase contrast image in the left panel; overlay image in the middle and image in the right panel shows fluorescence channel. (A) images showing Immobeads 150P treated with soluble mCherry and absence of fluorescence. (B) after functionalisation with mCherryP4o, Immobeads 150P displayed the presence of mCherryP4o aggregates as fluorescent (red) rings.

[0109] Figure 22 shows FESEM images of Immobeads 150P. (A) beads treated with soluble mCherry, images showing intact structure of beads which retained their smooth surface. (B) mCherryP4o- functionalised beads bearing aggregates embedded on their surface as patches (circled).

[0110] Figure 23 shows CLSM images of Bio-beads. (A) beads treated with soluble mCherry, images showing intact structure of beads with negligible uptake of soluble mCherry. (B) mCherryP4o- functionalised beads bearing aggregates embedded on their surface as patches (red fluorescence).

[0111] Figure 24 shows relative enzyme activities of PP fibres co-functionalised with BactAmyP4o (dark grey) and BactXylBP4o (light grey) enzymes. Fibres were assessed for 5 consecutive cycles. The error bars represent the standard deviations from triplicate experiments.

[0112] Figure 25 shows functionalisation efficiency with respect to amount of protein (%) retained after functionalisation of different matrices with BactAmyP4o (light grey) and mCherryP4o (dark grey). The error bars represent the standard deviations from triplicate experiments.

[0113] Figure 26 shows retained BactAmyP4o enzyme activity of different functionalised matrices. The activity is expressed in relative to the intact BactAmyP4o activity (considered it as 100%). The %functionalisation efficiency (line) showing the retained protein content of respective BactAmyP4o-functionalised matrices. The error bars represent the standard deviations from triplicate experiments.

[0114] Figure 27 shows CLSM images of bare and functionalised synthetic PP fibre (different regions) with BactAmyP4o. Phase contrast image in the left panel; overlay image in the middle and right panel is fluorescence channel. (A) PP fibre treated with SYPRO orange dye and (B) BactAmyP4o- functionalised PP fibre after stained with SYPRO orange dye.

[0115] Figure 28 shows reusability of BactAmyP4o-functionalised synthetic PP fibre at 70°C and 80°C. The error bars represent the standard deviations from triplicate experiments.

[0116] Figure 29 shows reusability of BactAmyP4o-functionalised BioCradle at 80° C. The error bars represent the standard deviations from triplicate experiments.

[0117] Figure 30 shows reusability of BactXylBP4o-functionalised synthetic PP fibre at 50° and 40°C. The error bars represent the standard deviations from triplicate experiments.

[0118] Figure 31 shows reusability of free aGPP4o and aGPP4o-functionalised CBSP097. Assays were performed at 70°C. The error bars represent the standard deviations from triplicate experiments.

[0119] Figure 32 shows reusability of free PGMP4o and PGMP4o-functionalised CBSP097. Assays were performed at 70°C. The error bars represent the standard deviations from triplicate experiments.

[0120] Figure 33 shows reusability of free PGiP4o and PGiP4o-functionalised CBSP097. Assays were performed at 70°C. The error bars represent the standard deviations from triplicate experiments.

[0121] Figure 34 shows reusability of free PGPP4o and PGPP4o-functionalised CBSP097. Assays were performed at 70°C. The error bars represent the standard deviations from triplicate experiments.

[0122] Figure 35 shows multiple sequence alignment of p40 proteins. Top line is p40 domain of ManA from Caldibacillus cellulovorans. This sequence is aligned against p40 from Phytohabitans suffuscus (Psuff), the p40 from Kibdelosporangium aridum (Karid), and the p40 from Archangium lipolyticum (Alipol).

[0123] Figure 36 shows relative activity of BactAmyP4o-functionalised CBSP097 matrices stored at 4°C (A) and 25°C (B). Figure 37 shows relative solubilising efficiency (%) of different reagents on mCherryP4o

[0124] Figure 38 shows FESEM images of non-functionalised and functionalised CBSP097 with p40 and three p40-homologs. Images are shown at 500x magnification (top panel) and 1000x magnification (bottom panel).

[0125] Figure 39 shows CLSM images of bare and functionalised CBSP097 with different p40- homologs. Fluorescence channel in the left panel; Phase contrast image in the middle and overlay image in the right panel is. (A) bare CBSP097 treated with SYPRO orange dye and (B)- (E) p40 and p40-homologs-functionalised CBSP097 after stained with SYPRO orange dye.

[0126] Figure 40 shows ATR-FTIR absorption spectrum of bare and functionalised CBSP097 matrices showing characteristic spectral bands.

[0127] Figure 41 shows reusability (over 10 consecutive cycles) of free F6PEP4o and F6PEP4o-CBSPO97. Assays were performed at 70°C. The error bars represent the standard deviations from triplicate experiments.

[0128] Figure 42 shows reusability (over 10 consecutive cycles) of free T4EP4o and T4EP4o-CBSPO97. Assays were performed at 70°C. The error bars represent the standard deviations from triplicate experiments.

[0129] Figure 43 shows the SpinChem® and its rotating bed reactor (RBR) compartments loaded with T4EP4o-functionalised PP fibre.

[0130] Figure 44 shows reusability and conversion rate of the reaction catalysed by T4E cofunctionalised PP fibre over 10 consecutive cycles.

[0131] Figure 45 representative chromatogram showing the separation of D-fructose (substrate) and D- tagatose (product) afterthe T4EP4o catalysed reaction.

[0132] Figure 46 shows the product titre of intermediate and final products from multi-enzyme reactions mediated by free enzymesP4o and enzymesP4o-functionalised CBSP097. Figure 47 shows representative plot indicating hot spots in the p40 sequence with higher aggregation propensity predicted from two different computational tools (A) tango and (B) AGGRESCAN.

[0133] Figure 48 shows SDS-PAGE profile showing distribution of expressed fusion protein. The samples were diluted 1 :1 with 2X Laemmli sample buffer and 10 pL of each sample was loaded onto the gel and stained with SimplyBlue™ SafeStain. Lane M, molecular weight marker; TP, Total protein; TS, Total soluble; Tl, Total insoluble. (A) mCherryP4o, (B) BactAmyP4o, (C) aGPP4o and (D) PGPP4o.

[0134] Figure 49 SDS-PAGE gel profile of different p40_IBs after purification. The samples were diluted 1 :1 with 2X Laemmli sample buffer and 10 pL of each sample was loaded onto the gel and stained with SimplyBlue™ SafeStain. (A) shows the mCherry fusion to different p40 deletion variants. Lane M, molecular weight marker; 1 , mCherryP4o_full (~50.6 kDa); 2, mCherryP4o_minus 1 (~48.2 kDa); 3, mCherryP4o_minus 2 (~46.1 kDa); 4, mCherryP4o_minus 3 (~39.5 kDa); 5, mCherryP4o_minus 4 (~37.9 kDa); 6, mCherryP4o_minus 5 (~33.5 kDa); 7, mCherryP4o_minus 6 (~31 kDa); (B) shows the BactAmy fusion to different p40 deletion variants. Lane M, molecular weight marker; 1 , BactAmyP4o_full (~79.4 kDa); 2, BactAmyP4o_minus 1 (~77 kDa); 3, BactAmyP4o_minus 2 (~74.8 kDa); 4, BactAmyP4o_minus 3 (~68.2 kDa); 5, BactAmyP4o_minus 4 (~66.7 kDa); 6, BactAmyP4o_minus 5 (~62.2 kDa); 7, BactAmyP4o_minus 6 (~59.7 kDa). (C) shows the aGP fusion to different p40 deletion variants. Lane M, molecularweight marker; 1 , aGPP4o_full (~89.2 kDa); 2, aGPP4o_minus 3 (~78.1 kDa); 3, aGPP4o_minus 4 (~76.5 kDa). (D) shows the PGP fusion to different p40 deletion variants. Lane M, molecular weight marker; 1 , PGPP4o_full (~47.9 kDa); 2, PGPP4o_minus 3 (~36.7 kDa); 3, PGPP4o_minus 4 (~35.1 kDa).

[0135] Figure 50 shows expression kinetics of mCherry fused to different p40 deletion variants produced in E. coli Tuner (DE3) cells upon different inducer concentrations.

[0136] Figure 51 shows (A) SDS-PAGE analysis of whole cells expressing mCherry fused to different p40 deletions variants. Cells collected at specific interval oftime were diluted 1 :1 with 2X Laemmli sample buffer and 10 pL of each sample was loaded onto the gel and stained with SimplyBlue™ SafeStain. The arrow indicates the protein band corresponding to soluble mCherry (~26.7 kDa). (B) Image showing fluorescence of total soluble and total insoluble fraction after cell lysis of respective mCherry p40 deletion fusion. Figure 52 shows confocal microscopy images of Tuner (DE3) cells expressing mCherry fused to different p40 deletion variants. Cells producing mCherry IBs fused to p40 deletion variants, (A) full, minus 1 , minus 2, minus 3 and (B) minus 4, minus 5 and minus 6.

[0137] Figure 53 shows total protein content of different purified IBs produced from various p40 deletion fusion proteins. (A) protein content (%) of mCherryP4o deletion mutants and BactAmyP4o deletion mutants, and (B) protein content (%) aGPP4o deletion mutant and PGP p40 deletion mutants.

[0138] Figure 54 shows field emission scanning electron microscopy (FESEM) images of purified mCherryP4o IBs produced from various p40 deletion fusion. Images showing mCherryP4o IBs produced by fusing, (A) p40_full, (B) p40_minus 1 , (C) p40_minus 2, (D) p40_minus 3, (E) p40_minus 4, (F) p40_minus 5 and (G) p40_minus 6.

[0139] Figure 55 shows field emission scanning electron microscopy (FESEM) images of purified BactAmyP4o IBs produced from various p40 deletion fusion. Images showing BactAmyP4o IBs produced by fusing, (A) p40_full, (B) p40_minus 1 , (C) p40_minus 2, (D) p40_minus 3, (E) p40_minus 4, (F) p40_minus 5 and (G) p40_minus 6.

[0140] Figure 56 shows field emission scanning electron microscopy (FESEM) images of purified aGPP4o and PGPP4o IBs produced from different p40 deletion fusion. Images showing aGPP4o IBs produced by fusing, (A) p40_full, (B) p40_minus 3, (C) p40_minus 4 and PGPP4o IBs produced by fusing (D) p40_fu II, (E) p40_minus 3, (F) p40_minus 4.

[0141] Figure 57 shows retained bio / catalytic activity of (A) mCherryP4o and (B) BactAmyP4o IBs produced by fusion of different p40 deletions.

[0142] Figure 58 shows retained catalytic activity of CBSP097 matrices functionalised with (A) BactAmyP4o, (B) aGPP4o and (C) PGPP4o IBs produced by fusion of different p40 deletions.

[0143] SEQ ID NO:1 amino acid sequence of p40 from beta-1 , 4-mannanase precursor Caldibacillus cellulovorans.

[0144] SEQ ID NO:2 amino acid sequence of p40 from Phytohabitans suffuscus (Psuff).

[0145] SEQ ID NO:3 amino acid sequence of p40 from Kibdelosporangium aridum (Karid).

[0146] SEQ ID NO:4 amino acid sequence of p40 from Archangium lipolyticum (Alipol).

[0147] SEQ ID NO:5 amino acid sequence of p40 and mCherry. SEQ ID NO:6 amino acid sequence of p40 and enhanced green fluorescence protein (eGFP).

[0148] SEQ ID NO:7 amino acid sequence of p40 and alpha-glucan phosphorylase (aGP).

[0149] SEQ ID NO:8 amino acid sequence of p40 and phosphoglucomutase (PGM).

[0150] SEQ ID NO:9 amino acid sequence of p40 and phosphoglucoisomerase (PGI).

[0151] SEQ ID NO:10 amino acid sequence of p40 and phosphoglycolatephosphatase (PGP).

[0152] SEQ ID NO:11 amino acid sequence of p40 and beta-galactosidase ( Gal).

[0153] SEQ ID NO:12 amino acid sequence of p40 and FTL (futalin).

[0154] SEQ ID NO:13 amino acid sequence of p40 and MVN (microvirin).

[0155] SEQ ID NO:14 amino acid sequence of p40 and metallothionein (metallo).

[0156] SEQ ID NO:15 amino acid sequence of p40 and anti-Hise nanobody (Anti-His).

[0157] SEQ ID NO:16 amino acid sequence of p40 and ZXR-2 (lytic peptide derived from N-terminal sequence of mauriporin, a scorpion venom toxin)

[0158] SEQ ID NO:17 amino acid sequence of p40 and BactXylB (xylanase from Bacillus subtilis).

[0159] SEQ ID NO:18 amino acid sequence of p40 and alpha amylase (BacAmy).

[0160] SEQ ID NO:19 amino acid sequence of p40 and fructose 6-phosphate 4-epimerase (F6PE).

[0161] SEQ ID NO:20 amino acid sequence of p40 and tagatose-4-epimerase (T4E).

[0162] SEQ ID NO:21 amino acid sequence of p40 mutant designated minus 1 .

[0163] SEQ ID NO:22 amino acid sequence of p40 mutant designated minus 2.

[0164] SEQ ID NO:23 amino acid sequence of p40 mutant designated minus 3.

[0165] SEQ ID NO:24 amino acid sequence of p40 mutant designated minus 4.

[0166] SEQ ID NO:25 amino acid sequence of p40 mutant designated minus 5.

[0167] SEQ ID NO:26 amino acid sequence of p40 mutant designated minus 6.

[0168] SEQ ID NO:27 amino acid sequence of peptide from endo-1 ,4-beta-xylanase of Kallotenue papyrolyticum (WP_161668714.1 ) .

[0169] SEQ ID NO:28 amino acid sequence of peptide from lytic polysaccharide monooxygenase

[0170] Kibdelosporangium aridum (WP_235039209.1).

[0171] SEQ ID NO:29 amino acid sequence of peptide from lytic polysaccharide monooxygenase

[0172] Kibdelosporangium aridum (WP_033383782.1).

[0173] SEQ ID NQ:30 amino acid sequence of peptide from lytic polysaccharide monooxygenase

[0174] Cystobacter fuscus (WP_002632296.1 ) .

[0175] SEQ ID NO:31 amino acid sequence of peptide cellulose-binding protein Kibdelosporangium aridum (RSM65244.1).

[0176] SEQ ID NO:32 amino acid sequence of peptide lytic polysaccharide monooxygenase Kibdelosporangium aridum (WP_037268603.1).

[0177] SEQ ID NO:33 amino acid sequence of peptide lytic polysaccharide monooxygenase Archangium violaceum (WP_204219339.1). SEQ ID NO:34 amino acid sequence of peptide lytic polysaccharide monooxygenase Archangium lipolyticum (WP_257447283.1).

[0178] SEQ ID NO:35 amino acid sequence of peptide lytic polysaccharide monooxygenase Cystobacter fuscus (WP_395837646.1 ) .

[0179] SEQ ID NO:36 amino acid sequence of peptide lytic polysaccharide monooxygenase Cystobacter fuscus (WP_095989672.1 ) .

[0180] SEQ ID NO:37 amino acid sequence of peptide lytic polysaccharide monooxygenase

[0181] Kibdelosporangium banguiense (WP_245378406.1).

[0182] SEQ ID NO:38 amino acid sequence of peptide lytic polysaccharide monooxygenase Cystobacter fuscus (WP_395850959.1 ) .

[0183] SEQ ID NO:39 amino acid sequence of peptide lytic polysaccharide monooxygenase Cystobactersp. (HEX8536417.1).

[0184] SEQ ID NO:40 amino acid sequence of peptide lytic polysaccharide monooxygenase Vitiosangium sp. GDMCC 1.1324 (WP_108066648.1).

[0185] SEQ ID NO:41 amino acid sequence of peptide lytic polysaccharide monooxygenase

[0186] Cystobacter ferrugine us (WP_071897161 .1).

[0187] SEQ ID NO:42 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Actinoalloteichus spitiensis (WP_245533458.1).

[0188] SEQ ID NO:43 amino acid sequence of peptide lytic polysaccharide monooxygenase Cystobactersp. (MET0403368.1).

[0189] SEQ ID NO:44 amino acid sequence of peptide lytic polysaccharide monooxygenase Euzebyales bacterium (HSK96568.1)

[0190] SEQ ID NO:45 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Actinopolyspora xinjiangensis (WP_211481187.1).

[0191] SEQ ID NO:46 amino acid sequence of peptide lytic polysaccharide monooxygenase Antribacter soli (WP_236091496.1).

[0192] SEQ ID NO:47 amino acid sequence of peptide cell wall protein Actinoalloteichus sp. AHMU CJ021 (AUS77393.1).

[0193] SEQ ID NO:48 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Actinoalloteichus (WP_081715513.1).

[0194] SEQ ID NO:49 amino acid sequence peptide lytic polysaccharide monooxygenase Actinokineospora soli (GAA4422109.1).

[0195] SEQ ID NO:50 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Qaidamihabitans albus (WP_235999690.1).

[0196] SEQ ID NO:51 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Prauserella shujinwangii (WP_245900408.1). SEQ ID NO:52 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Actinopolyspora erythraea (WP_043573225.1).

[0197] SEQ ID NO:53 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Actinopolyspora mortivallis (WP_106113552.1).

[0198] SEQ ID NO:54 amino acid sequence of peptide lytic polysaccharide monooxygenase

[0199] Cellulomonas chengniuliangii (WP_227584873.1).

[0200] SEQ ID NO:55 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Actinopolyspora mortivallis (WP_019853974.1).

[0201] SEQ ID NO:56 amino acid sequence of peptide lytic polysaccharide monooxygenase

[0202] Cellulomonas chengniuliangii (WP_227569922.1).

[0203] SEQ ID NO:57 amino acid sequence of peptide lytic polysaccharide monooxygenase

[0204] Actinokineospora guangxiensis (WP_378247683.1).

[0205] SEQ ID NO:58 amino acid sequence of peptide lytic polysaccharide monooxygenase

[0206] Archangium (WP_395805807.1).

[0207] SEQ ID NO:59 amino acid sequence of peptide lytic polysaccharide monooxygenase

[0208] Actinokineospora sp. UTMC 2448 (WP_258903647.1).

[0209] SEQ ID NQ:60 amino acid sequence of peptide lytic polysaccharide monooxygenase

[0210] Actinokineospora fastidiosa (WP_229786899.1).

[0211] SEQ ID NO:61 amino acid sequence of peptide lytic polysaccharide monooxygenase

[0212] Streptomonospora nanhaiensis (MBV2365531 .1).

[0213] SEQ ID NO:62 amino acid sequence of peptide lytic polysaccharide monooxygenase

[0214] Streptomonospora nanhaiensis (WP_308118924.1).

[0215] SEQ ID NO:63 amino acid sequence of peptide lytic polysaccharide monooxygenase Antribacter gilvus (WP_206074568.1).

[0216] SEQ ID NO:64 amino acid sequence of peptide lytic polysaccharide monooxygenase

[0217] Streptomonospora mangrove (WP_270070392.1).

[0218] SEQ ID NO:65 amino acid sequence of peptide lytic polysaccharide monooxygenase

[0219] Archangium violaceum (WP_203405851 .1).

[0220] SEQ ID NO:66 amino acid sequence of peptide lytic polysaccharide monooxygenase

[0221] Cellulomonas cellasea (WP_289565354.1).

[0222] SEQ ID NO:67 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Goodfellowiella coeruleoviolacea (WP_253779939.1).

[0223] SEQ ID NO:68 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Actinoalloteichus hymeniacidonis (WP_069848685.1).

[0224] SEQ ID NO: 69 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Actinopolyspora sp. H202 (MGJ7906582.1). SEQ ID NO:70 amino acid sequence of peptide hypothetical protein GCM10027444_06860 Actinopolyspora lacussalsi (GAB3545420.1).

[0225] SEQ ID NO:71 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Actinopolyspora alba group (WP_092926416.1).

[0226] SEQ ID NO:72 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Cellulomonas timonensis (WP_082812676.1).

[0227] SEQ ID NO:73 amino acid sequence of peptide lytic polysaccharide monooxygenase Allostreptomyces psammosilenae (WP_218903958.1).

[0228] SEQ ID NO:74 amino acid sequence of peptide lytic polysaccharide monooxygenase Salinactinospora qingdaonensis (WP_344975556.1).

[0229] SEQ ID NO:75 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Actinopolyspora mzabensis (WP_092626937.1).

[0230] SEQ ID NO:76 amino acid sequence of peptide lytic polysaccharide monooxygenase Nocardiopsis ansamitocini (WP_285759904.1).

[0231] SEQ ID NO:77 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Allostreptomyces psammosilenae (WP_218903992.1).

[0232] SEQ ID NO:78 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Cellulomonas sp. NS3 (WP_258725492.1).

[0233] SEQ ID NO:79 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Cellulomonas cellasea (WP_084142425.1).

[0234] SEQ ID NO:80 amino acid sequence of peptide lytic polysaccharide monooxygenase Nocardiopsis trehalosi (WP_245646357.1).

[0235] SEQ ID NO:81 amino acid sequence of peptide lytic polysaccharide monooxygenase Thermobifida halotolerans (WP_084012808.1).

[0236] SEQ ID NO:82 amino acid sequence of peptide hypothetical protein Q760_14795 Cellulomonas cellasea DSM 20118 (KGM03715.1).

[0237] SEQ ID NO:83 amino acid sequence of peptide putative carbohydrate-binding protein with CBM5 and CBM33 domain Thermobifida halotolerans (MEY9212146.1).

[0238] SEQ ID NO:84 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Allostreptomyces psammosilenae (WP_218904110.1).

[0239] SEQ ID NO:85 amino acid sequence of peptide lytic polysaccharide monooxygenase Nocardiopsis trehalosi (XVZ52746.1).

[0240] SEQ ID NO:86 amino acid sequence of peptide lytic polysaccharide monooxygenase Thermobifida cellulosilytica (WP_232306641 .1).

[0241] SEQ ID NO:87 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Actinoalloteichus (WP_075740328.1). SEQ ID NO:88 amino acid sequence of peptide cellulose-binding protein Thermobifida cellulosilytica TB100 (KUP97844.1).

[0242] SEQ ID NO:89 amino acid sequence of peptide cellulose-binding protein Marinitenerispora sediminis (RCV49493.1).

[0243] SEQ ID NO:90 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Promicromonospora sp. CA-294202 (XVZ32663.1).

[0244] SEQ ID NO:91 amino acid sequence of peptide lytic polysaccharide monooxygenase Marinitenerispora sediminis (WP_199493194.1).

[0245] SEQ ID NO:92 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Cellulomonas sp. 179-A 4D5 NHS (WP_412871499.1).

[0246] SEQ ID NO:93 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Promicromonospora sp. CA-289581 (XWA39973.1).

[0247] SEQ ID NO:94 amino acid sequence of peptide lytic polysaccharide monooxygenase Kribbella sp. CA-294648 (WP_433020383.1).

[0248] SEQ ID NO:95 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Cellulomonas cellasea (WP_183296256.1).

[0249] SEQ ID NO:96 amino acid sequence of peptide lytic polysaccharide monooxygenase Micromonospora sp. NBC_01813 (WP_326553736.1).

[0250] SEQ ID NO:97 amino acid sequence of peptide cellulose-binding protein Lentzea indica (NKE62969.1).

[0251] SEQ ID NO:98 amino acid sequence of peptide cellulose-binding protein Kribbella albertanoniae (TDC14488.1).

[0252] SEQ ID NO:99 amino acid sequence of peptide lytic polysaccharide monooxygenase Lentzea indica (WP_223165838.1).

[0253] SEQ ID N0:100 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Saccharomonospora saliphila (WP_028661718.1).

[0254] SEQ ID NO:101 amino acid sequence of peptide lytic polysaccharide monooxygenase Kribbella sp. NPDC023855 (WP_360281864.1).

[0255] SEQ ID NO:102 amino acid sequence of peptide lytic polysaccharide monooxygenase Lentzea indica (XWG55099.1).

[0256] SEQ ID NO:103 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Kribbella albertanoniae (WP_238176799.1).

[0257] SEQ ID NO:104 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Actinoalloteichus hoggarensis (WP_245856695.1).

[0258] SEQ ID NO:105 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Promicromonospora sp. CA-293590 (XVZ18152.1). SEQ ID NO:106 amino acid sequence of peptide lytic polysaccharide monooxygenase Micromonospora sp. NPDC049679 (WP_357428893.1).

[0259] SEQ ID NO:107 amino acid sequence of peptide cellulose-binding protein Streptomonospora sp.

[0260] PA3 (MUL42669.1).

[0261] SEQ ID NO:108 amino acid sequence of peptide lytic polysaccharide monooxygenase Solwaraspora sp. WMMD1047 (WP_278094743.1).

[0262] SEQ ID NO:109 amino acid sequence of peptide lytic polysaccharide monooxygenase Kribbella sp. CA-291356 (XVZ00809.1).

[0263] SEQ ID NO:110 amino acid sequence of peptide lytic polysaccharide monooxygenase Streptomonospora sp. PA3 (WP_343233562.1).

[0264] SEQ ID NO:111 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein, partial Actinoalloteichus caeruleus (WP_030107472.1).

[0265] SEQ ID NO:112 amino acid sequence of peptide lytic polysaccharide monooxygenase Pilimelia sp. (HYN94230.1).

[0266] SEQ ID NO: 113 amino acid sequence of peptide lytic polysaccharide monooxygenase Solwaraspora sp. WMMD406 (WP_278120201 .1).

[0267] SEQ ID NO:114 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Promicromonospora sp. CA-289599 (WP_432895007.1).

[0268] SEQ ID NO:115 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Promicromonospora panici (WP_207211759.1).

[0269] SEQ ID NO:116 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Kribbella sp. NPDC056345 (WP_376027776.1).

[0270] SEQ ID NO:117 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Nocardiopsis mangrovi (WP_378572488.1).

[0271] SEQ ID NO:118 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Promicromonospora sp. CA-294714 (XVZ55973.1).

[0272] SEQ ID NO:119 amino acid sequence of peptide lytic polysaccharide monooxygenase Kribbella catacumbae (WP_202806718.1).

[0273] SEQ ID NO:120 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Promicromonospora sp. NPDC050249 (WP_357511103.1).

[0274] SEQ ID NO:121 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Promicromonospora sp. NPDC060271 (WP_377224174).

[0275] SEQ ID NO:122 amino acid sequence of peptide lytic polysaccharide monooxygenase auxiliary activity family 9 protein Nocardiopsis sediminis (WP_378533207.1).

[0276] SEQ ID NO:123 amino acid sequence of peptide lytic polysaccharide monooxygenase Thermobifida alba (HLU95482.1). SEQ ID NO:124 amino acid sequence of peptide lytic polysaccharide monooxygenase Lentzea flava (WP_253730525.1).

[0277] SEQ ID NO:125 amino acid sequence of linker.

[0278] Detailed Description

[0279] General Techniques and Definitions

[0280] Unless otherwise indicated, any recombinant molecular biology or immunological techniques described herein are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1- 4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0281] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both means or for either meaning. Furthermore, a list or features including the phrase “and / or” between the second last and last feature means that any one or more of the listed features may be present in any combination.

[0282] Reference to the singular forms “a”, “an” and “the” is also understood to imply the inclusion of plural forms unless the context dictates otherwise.

[0283] As used in this application, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or.” That is, unless specified otherwise, or clear from context, “X employs A or B” is intended to mean any ofthe natural inclusive permutations. That is, if X employs A; X employs B; or X employs both A and B, then “X employs A or B” is satisfied under any of the foregoing instances. Further, at least one of A and B and / orthe like generally means A or B or both A and B.

[0284] As used herein, the term “about”, unless stated to the contrary, refers to + / - 10%, more preferably + / - 5%, of the designated value.

[0285] The term “particle” as used herein refers to a substantially insoluble entity consisting of a protein. These entities may be spherical, ellipsoidal, in string form, in sheets, discs or any other shape. The particles may be of any size between 1 nm and 100 pm.

[0286] The term “protein” as used herein means a polymer made up of amino acids linked together by peptide bonds, and includes fragments or analogues thereof. The terms “polypeptide” and “protein” are used interchangeably herein, although for the purposes of the present invention a “polypeptide” may constitute a portion of a full length protein or a complete full length protein.

[0287] The term “substantially” as used herein means the majority but not necessarily all, and thus in relation to a modified polypeptide “substantially” lacking a component region of a corresponding wild-type polypeptide, the modified polypeptide may retain a portion of that component region. For example, a modified polypeptide “substantially” lacking a component region of a corresponding wild-type polypeptide may retain approximately 50 percent or less of the sequence of the component region, although typically the component region is rendered structurally and / or functionally inactive by virtue of the proportion of the sequences of the region omitted.

[0288] The term “affinity separation” as used herein refers to a method of separating, purifying, removing, enriching and / or concentrating a component from a mixture or suspension.

[0289] The terms “absorbed, combined, bound to, or contained within the porous matrix” as used herein encompasses any covalent or non-covalent interaction. Examples include pi-pi stacking, hydrogen bonds, ionic interactions, hydrophobic interactions, and van der Walls forces. Examples also include direct binding.

[0290] The term “target compound” is understood to a carbohydrate, a bacterium, a protein or a metal. As used herein the term target compound may be used interchangeably with the term “target component”.

[0291] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0292] As used herein, the term “identity” means the percentage of identical amino acid residues at corresponding portions in two or more sequences when sequences are aligned to maximise sequence matching, i.e. taking into account gaps and insertions. Identity can be readily calculated using known methods, including, but not limited to those described in Computational Molecular Biology, Lesk AM ed. Oxford University Press New York, 1988; Computer Analysis of Sequence data, Part I Griffin AM and Griffin HG eds., Humana Press, New Jersey, 1994; Sequence analysis in molecular biology, von Heinje G, Academic Press, New Jersey, 1994). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determined identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequence include, but are not limited to, the GCG program package, BLASTP, BLASTN and FASTA. The well-known Smith Waterman algorithm may also be used to determine identity.

[0293] The p40 protein domain was initially identified as an unknown domain within the multidomain - mannanase enzyme (ManA) from Caldibacillus cellulovorans (Sunna, A. et al., (2000) Applied and Environmental Microbiology, 66(2), 664-670). Previous attempts to produce p40 in Escherichia coli led to the formation of insoluble inclusion bodies. Recently, sequence analysis classified this domain as a lytic polysaccharide monooxygenase (LPMO), specifically belonging to Auxiliary Activity Family 10 (AA10) in the CAZy database (www.cazy.org / AA10). The generation of p40-PNPs has been previously described in US 2012 / 0009624, the entire contents of which are herein incorporated by reference.

[0294] LPMOs are copper-dependent enzymes catalysing the oxidative cleavage of glycosidic bonds in polysaccharides like cellulose and chitin. The AA10 family include enzymes derived from archaea, bacteria, fungi, or viruses. LPMOs are often associated with carbohydrate-binding modules or glycoside hydrolase domains. Their catalytic domain typically comprises an immunoglobulin-like p-sandwich structure with a copper ion coordinated by two conserved histidines (Vaaje-Kolstad, G. et al., (2017) Current Opinion in Structural Biology, 44, 67-76, Span, E. A. et al., (2015) Current Opinion in Structural Biology, 35, 93-99).

[0295] The inventors investigated the ability of an aggregating partner (domain p40) to produce functional / biologically active self-assembling protein particles. They show that domain p40 displayed reversible solubilisation-reaggregation properties and describe extensively how this property can be exploited as a fasts and facile platform to biofunctionalise various commercially available matrices. The proposed innovation has the potential to deliver a single strategy to immobilise / functionalise enzymes onto various supports without use of any chemical crosslinking agents. For example, using this approach the achieved cross-linker free surface functionalisation of inorganic materials including non-woven fabrics made of cellulose, and polyethylene terephthalate microfibers, cellulose and dextran microcarriers, polymethacrylate Immobeads 150P and polystyrene Bio-Beads with various p40 based self-assembling protein particles (enzyme). This creates a platform for operational immobilised enzyme reactors with simplistic and cost-efficient functionalisation strategy.

[0296] In one example, the p40 protein has one of the following activities selected from:

[0297] (i) Lytic xylan monooxygenase / xylan oxidase (C4-dehydrogenating) (EC 1.14.99.-);

[0298] (ii) Lytic chitin monooxygenase (EC 1.14.99.53);

[0299] (iii) Lytic cellulose monooxygenase (C1-hydroxylating) (EC 1.14.99.54); or

[0300] (iv) Lytic cellulose monooxygenase (C4-dehydrogenating) (EC 1 .14.99.56), wherein EC refers to Expasy reference (ww .enzyme.expasy.org).

[0301] The enzymes in the AA10 family were originally classified as chitin-binding proteins (CBM33). Vaaje-Kolstad, G. et al., (2017) supra have shown that these proteins are in fact oxidative enzymes. They are now reclassified in the AA category of CAZy. Of the 11404 entries in this family, only 30 enzymes have been experimentally characterized to have enzymatic activity.

[0302] In a particular example, the p40 protein comprises a sequence derived from ManA of C. celulovorans. In a further example, the p40 protein comprises or consists of the sequence set forth in SEQ ID NO:1 or homolog thereof comprising a sequence at least 60% identical thereto. In one example, the p40 protein is PsufP4o which is the predicted chitin-binding protein of Phytohabitans suffuscus having GenBank reference BCB91526, or lytic polysaccharide monooxygenase of Phytohabitans suffuscus with reference WP_232075698 (non-redundant protein record on NCBI). The PsufP4o sequence is set forth in SEQ ID NO:2. In one example, the p40 protein is KariP4o which is the predicted cellulose-binding protein of Kibdelosporangium aridum having GenBank reference RSM65244, or lytic polysaccharide monooxygenase of Kibdelosporangium aridum with reference WP_037268603. The KariP4o sequence is set forth in SEQ ID NO:3. In one example, the p40 protein is AlipP4o which is the predicted lytic polysaccharide monooxygenase of Archangium lipolyticum with reference WP_257447283. The AlipP4o sequence is set forth in SEQ ID NO:4.

[0303] A sequence alignment of representative p40 sequences is shown in Figure 35.

[0304] Functional Inclusion bodies

[0305] Inclusion bodies (IBs) are dense, spherical, aggregated proteins, mostly formed in the cytoplasm of prokaryotes due to the overexpression of native or heterologous proteins. A number of factors are believed to contribute to the production of inclusion bodies, but the primary belief is a mismatch between the rate of protein production and the rate of protein misfolding within the prokaryote (e.g. E. coli) cytoplasm. This mismatch overwhelms the E. coli quality control system, resulting in insoluble aggregated protein. Over the past decade, research has revealed that IBS are formed by a mixture of amyloid protein and 70-95% recombinant proteins with native confirmation. Several IBs formed by critical human proteins including the enzymes dihydrofolate reductase (DHFR) and catalase (CAT), the growth factor leukemia inhibitory factor (LIF), and the chaperon Hsp70 have resulted in positive physiological impact to mammalian cells, exhibiting significant enhancement of cell survival and / or proliferation under stress conditions and inhibition of apoptosis.

[0306] Unlike traditional bacterial inclusion bodies (IBs), which were viewed as inert protein aggregates requiring complex refolding to restore protein functionality, active or functional inclusion bodies (FIBs) retain significant biological activity within their nanostructured particles. This paradigm shift has positioned FIBs as versatile biomaterials with applications spanning drug delivery, tissue engineering, biocatalysis, and biosensing. The unique properties of FIBs such as high protein yield, stability, simplified purification processes, tunable morphology, and the ability to encapsulate functional proteins have unlocked their potential across diverse fields, redefining their role from waste products to high-value materials. Fusion strategies using aggregationinducing or pull-down tags offer a promising approach to produce FIBs recombinantly in bacteria. These tags, typically a short polypeptide sequences or domains, promote controlled protein aggregation while preserving biological activity, yielding FIBs. The selection of suitable fusion tag often begins with computational methods that leverage understanding of protein aggregation and folding. Several powerful predictive algorithms like AGGRESCAN, A3D, Tango, AggreProt and Deepcoil aid in analysing the structure and sequence characteristics of potential fusion tags to assess their intrinsic aggregation propensity. In addition to computational predictions, several key factors play a critical role in selecting tags for effective FIBs production. The primary concern is the tag's biocompatibility and non-toxicity, ensuring strong protein expression while maintaining host cell viability. Additionally, ease of genetic fusion, and the absence of proteolytic cleavage sites within the tag are crucial for efficient cloning and stable expression. Specific aggregationinducing tags can effectively promote structured aggregation in certain proteins, leading to the formation of high performing FIBs with retained biological activity. However, these same tags may impose conformational constraints on other proteins, resulting in decreased or loss of activity. Although computational predictions are valuable, they rely on statistical models and estimates, which may not fully reflect the complexity of real biological interactions occurring in vivo. Due to the current limitations in understanding, it is not possible to accurately predict effective combinations of target enzymes, linkers, and aggregation-inducing tags.

[0307] Described herein are the controlled production of FIBs using a p40 fusion partner. P40 is a protein domain of 203 amino acids with a strong propensity for aggregation when expressed recombinantly in E. coli. Given that p40 fusions consistently enabled the production of high yield and stable FIBs of diverse bioactive proteins and enzymes with different complexity, the inventors further explored sequence determinants within p40 to obtain shorter aggregation-inducing tags capable of producing FIBs. Using two computational models, namely Tango and AGGRESCAN, several aggregation propensity regions or “hot spots’’ within p40 were identified. The computational prediction based on AGGRESCAN identified at least six possible regions with higher aggregation propensity. Based on the Tango algorithm, four major aggregation-inducing regions were identified each with a length of ~10-20 amino acids.

[0308] Six different truncated versions of p40 were generated from AGGRESCAN prediction, each designed by deleting specific “hot spots’’ from the C-terminus of p40 domain such that each truncation retained at least one predicted "hot spot". The efficacy ofthese truncated p40 variants as fusion tags for producing FIBs was then investigated. To achieve this, these six p40 deletion variants (designated as minus 1 , minus 2, minus 3, minus 4, minus 5, and minus 6) and p40_full (without deletion) were genetically fused to the C-termini of four target proteins exhibiting diverse structural complexities: a simple monomeric fluorescent protein (mCherry), a monomeric a- amylase (BactAmy), a homodimeric phosphoglycolate phosphatase (PGP), and a heterotetrameric a-glucan phosphorylase (aGP). The sequences of these p40 deletion mutants are shown in Table 4.

[0309] PNP proteins

[0310] The present disclosure relates to functionalised matrices comprising: (i) a protein nanoparticle (PNP) having an aggregating part capable of forming or aggregating into a protein particle and a functional part capable of binding to or being bound by a target compound; and (ii) a porous matrix; wherein the PNP is absorbed, combined, bound to or contained within the porous matrix.

[0311] The PNP is typically produced by recombinant DNA technology where nucleotide fragments encoding the desired proteins, peptides or fragments are joined together with or without an interspaced nucleotide fragment encoding a spacer or linker region. On part of the protein is preferably p40 or a homolog thereof, or any other protein such as alpha-amylase, human alpha-fetoprotein, Somatotropin, cellulose binding domain from Clostridium, or other proteins such as synthetic proteins or peptides, which forms or aggregates into suitable particles when expressed in an appropriate host organism such as Escherichia coli. At least one other part of the PNP may comprise an antibody binding domain such as protein A, protein G, protein L, or a single chain antibody, avidin, streptavidin, an enzyme, an inhibitor, an antigenic determinant, an epitope, a binding site, a lectin, a polyhistidine, an oligohistidine, a receptor, a hormone, a signalling molecule, a polypeptide with specific or group specific binding capabilities, or any combination thereof.

[0312] Uses of the functionalised matrices

[0313] The functionalised matrices of the present disclosure have particular utility in affinity separation. In some examples, the matrix is spherical or substantially spherical. In some examples, the matrix is a fibrous material. Imaging analysis of the functionalised matrices show that after functionalisation, the reaggregated p40 nanoparticles are uniformly distributed through the matrix material. In some embodiments, the matrix is co-functionalised with different p40 PNPs.

[0314] One advantage of the functionalised matrix is that they are reusable. The experimental data shown herein demonstrated that enzymatic activity of an enzyme- p40 PNP is maintained over repeated use. In some examples, the enzymatic activity is maintained at greater than 50% over 10 cycles. The functionalised matrices may be used to separate a target component present in a sample, such as a biological sample, or a non-biological sample. The sample may be for example, blood, blood plasma, blood serum, blood derived precipitates or supernatants, animal extracts or secretions, milk, colostrum, whey orany other milk derived product orfraction thereof, fermentation broths, liquids or fractions thereof, cell lysates, cell culture supernatants, cell extracts, cell suspensions, viral cultures or lysates, plant extracts or fractions thereof.

[0315] Binding of the target component to the functionalised matrix allows the target component to be separated from the mixture. The target molecules can, if desired, be separated from the affinity matrix by elution through methods well known to persons skilled in the art.

[0316] The target component may be recovered from the functional matrix to which the target component is bound, and this recovery may involve at least one elution step. In this regard, the relevant eluant(s) may comprise a solution with compounds imparting high or low pH, high or low salt concentrations or compounds with competitive binding capacity. Such solutions can comprise inorganic or organic acids or salts thereof, chaotropic salts, or compounds with competitive binding capacity. For example, a buffer comprising glycine adjusted to a pH in the range of about 1.5 to 4. Other examples include buffers comprising citric, acetic, succinic, lactic, tartric, formic, propionic, boric or phosphoric acids orsalts thereof. The eluant may also comprise a solution of one or more inorganic acids, for example hydrochloric acid, sulphuric acid and nitric acid, or salts thereof such as sodium chloride, potassium chloride, ammonium chloride, sodium sulphate, potassium sulphate or ammonium sulphate. The eluant may also comprise a solution of one or more organic or inorganic basic compounds or salts thereof such as methylamine, piperazine, carbonate, phosphate, borate or ammonium hydroxide. The eluant may also comprise chaotropic compounds such as urea, guanidine, potassium iodide, sodium iodide, thiocyanates, detergents, hydrophobic molecules such as organic solvents, or any other molecule capable of weakening, breaking or disrupting molecular structures or bonds.

[0317] The eluant(s) used in the elution step(s) may have a pH in the range of about 1 .0 to about 14.0. The eluants may have ionic strengths in the range from about 1 x10“3to about 25.

[0318] Kits

[0319] The present disclosure also provides kits for separating, purifying, removing, enriching and / or concentrating a component from a mixture or suspension. Typically, kits for carrying out a method of affinity separation contain at least a number of the reagents required to carry out the method. Typically, the kits of the disclosure will comprise one or more containers, containing for example, matrices, wash reagents, and / or other reagents capable of releasing a bound component from a polypeptide or fragment thereof. Typically, a kit of the present disclosure will also include instructions for using the kit components to conduct the appropriate methods.

[0320] Methods and kits of the present disclosure find application in any circumstance in which it is desirable to purify any component from any mixture. The kits may also be used in in vitro diagnostic applications, tissue regeneration (e.g. such as treatment of burns), wound healing and organoid culture.

[0321] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0322] EXAMPLES

[0323] Materials and Methods

[0324] Chemicals and reagents

[0325] All chemical and reagents used in this study were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia), unless stated otherwise.

[0326] Plasmid construction, bacterial strains and culture conditions

[0327] All molecular biology procedures were performed as described by Russell and Sambrook (Sambrook, J., Russell, D.W., Cold Spring Harbor Laboratory. (2001) Molecular cloning: a laboratory manual 3rd edition. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory).

[0328] Inserts of mCherry (UniProt: X5DSL3), eGFP (UniProt: P42212), a-amylase (Uniprot: C6KML4), p-xylanase (Uniprot: Q45VU1), a-glucan phosphorylase (Uniprot: B8E0Q6), phosphoglucomutase (Uniprot: Q5SKJ3) and glucose-6-phosphate isomerase / phosphoglucoisomerase (Uniprot: Q5SLL6), tagatose 4-epimerase (Uniprot: A0A662GG58) and fructose 6-phosphate 4-epimerase (Uniprot: H1XRG1) were synthesised as codon optimised gBIock Gene Fragments (Integrated DNA Technologies, Singapore) for expression in Escherichia coli Tuner® (DE3) cells (Novagen, EMD Millipore, Billerica, MA, USA). The inserts contained Ncol and Hindi II sites for ligation into plasmid pETDuet-1 -p533 (a gift from Dr Moreland Gibbs). pETDuet-1-p533 harbours the gene sequence of the p40 domain of ManA from Caldibacillus cellulovorans preceded by (GGGGS)3 flexible linker sequence. The gBIock inserts of target proteins were ligated (4:1 insert: vector ratio) to Ncol and Hindi 11 linearised pETDuet-1 p533 a using the Quick Ligation Kit (NEB, USA) to obtain different p40 containing expression plasmids.

[0329] The different constructs are shown schematically in Table 2 below. The sequences of these constructs are shown in Table 3.

[0330] Bacterial transformation and screening

[0331] Bacterial transformations were performed as described in Novagen protocol TB009 Rev. F0104 (Novagen, USA). The ligated plasmids were propagated by transforming them into recA deficient NEB 5-alpha Competent E. co / / (NEB) and cultivations on Luria-Bertani (LB) agar plates supplemented with ampicillin (100pg / mL) as the selectable marker. Positive transformants were screened by colony polymerase chain reaction (PCR) with the OneTaq® DNA Polymerase kit (NEB) and T7-promoter and T7-terminator primers (IDT). The PCR amplification was with 10 min denaturation at 94°C followed by 30 cycles of denaturation at 94°C forthe 30s, annealing at 50°C for 30s, primer extension at 72° C for 1 min with a final extension at 72°C for 7 min. The amplicons were analysed by agarose gel electrophoresis using 1 % agarose gels in TAE buffer (40 mM Tris- acetate, 1 mM EDTA). DNA was visualised by the addition of GelRed® Nucleic Acid Gel Stain (Biotium, USA) and the Quick-Load® Purple 1 kb DNA Ladder (NEB) was used to determine the correct size of the PCR products. Plasmid purification was performed using the Monarch® Plasmid Miniprep Kit (NEB) according to the manufacturer’s protocol. Plasmids from positive NEB 5-alpha transformants were propagated and isolated from overnight cultivation at 37°C in

[0332] LB broth supplemented with ampicillin (1 OOpg / mL). The isolated plasmids were used to transform E. coli Tuner® (DE3) (Novagen) competent cells.

[0333] Table 2 Exemplified p40 constructs Table 3 Sequences of the exemplified p40 constructs

[0334] Construction of expression plasmids containing p40-deletion variants

[0335] All molecular biology procedures were performed as described by Russell and Sambrook (Sambrook, J., Russell, D.W., Cold Spring Harbor Laboratory. (2001) Molecular cloning: a laboratory manual 3rd edition. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory).

[0336] The plasmid pETDuet-1 p533, generously provided by Dr. Moreland Gibbs, served as the backbone for all constructs. This plasmid originally carried the p40 domain sequence of the multi-domain p-1 ,4-mannanase (ManA) from Caldibacillus cellulovorans (Sunna A, Gibbs MD, Chin CW, Nelson PJ & Bergquist PL (2000) Applied Environmental Microbiology, 66:664-670) followed by a short (GGGGS)3 flexible linker. Tables 4 and 5 schematically represent the amino acid sequences of p40 deletion variants, their respective restriction sites, and the final recombinant constructions. For cloning and plasmid propagation, NEB 5-alpha Competent Escherichia coli cells (New England Biolabs, Notting Hill, VIC, Australia) were used. Recombinant protein expression was carried out in E. coli Tuner (DE3) cells (Novagen, EMD Millipore, Billerica, MA, USA). All E. coli strains were cultured in Luria-Bertani (LB) medium supplemented with 100 pg / mL carbenicillin.

[0337] Table 4 Sequences of the p40 deletion variants

[0338] Table 5 Different fusions of p40 deletion variants.

[0339] GH; Glycoside Hydrolase, GT; GlycosylTransferase (www.cazy.org)

[0340] Recombination production of different p40 based inclusion bodies (IBs)

[0341] The production of different p40 IBs was started by raising the primary seeding culture at 37°C overnight with shaking (200 rpm). 2% of overnight grown seed culture was added to the fresh medium and incubated until the ODsoo reached 0.6-0.8 (mid-log phase). The target protein expression was initiated by adding 0.4 mM IPTG and the cultures were set to incubate at 37°C with shaking (250 rpm) for 6 h. Cells were harvested by centrifugation by 6000 x g for 20 min at 4°C. The collected cell pellets were washed with 1x Phosphate-buffered saline (PBS) pH 7.4 and the dry cell pellet was stored at -30°C until further use.

[0342] The production of all p40 deletion variants fused to mCherry, a-amylase (BactAmy), a- glucan phosphorylase (aGP) and phosphoglycolate phosphatase (PGP) followed the same experimental setup as described in the previous paragraph.

[0343] Purification of different p40 based IBs

[0344] Cell pellets were resuspended in 5% culture volume of lysis buffer (50 mM Tris-HCI, 100 mM NaCI, 1 mM EDTA, 1 mM serine protease inhibitor Pefabloc, and 1 % glycerol, pH 8). The resuspended cell pellets were homogenised by gentle vortexing followed by cell disruption by sonication (8 min with 5s on and 10s off duty cycle at 40% of the input amplitude) in a Branson 450 digital sonifier (Marshall Scientific, USA). The cell lysate was centrifuged at 12,000 x g for 15 min at 4°C to separate the insoluble pellet from the soluble fraction. The obtained insoluble pellet containing the target IBs was washed once with 1x PBS buffer and then treated with B- PER® (in phosphate buffer) reagent (Thermo Fisher Scientific).

[0345] Briefly, 1 g of the insoluble pellet was treated with 4 mL of B-PER supplemented with 5 units of Benzonase (Sigma-Aldrich) and incubated at room temperature for 15 min with gentle agitation. After incubation, the supernatant containing solubilised protein was separated from the insoluble pellet containing target p40 IBs by centrifugation at 12,000 x g for 15 min at 4°C. The pellet (insoluble protein fraction) obtained was washed twice with diluted B-PER (1 in 10 parts of MilliQ water). A final wash with 0.5% Triton X-100 was used before the sample was stored at - 30°C until further use.

[0346] Solubilisation and reaggreaation of o40-based IBs

[0347] Approximately 8 mg of each p40 IBs pellets were taken and solubilised using 200 pL of solubilisation buffer containing between 2 and 3 M guanidinium hydrochloride (GdmCI) and 5 mM DTT in 50 mM Tris-HCI, pH 8.5. Solubilisation of all IBs was performed for 1 h with gentle agitation at room temperature. Complete solubilisation was confirmed by measuring the turbidity at 350 nm in a BMG FLUOstar Omega after blanking the solution with respective concentrations of GdmCI. Solubilisation was further confirmed by the absence of a visible pellet after centrifugation ofthe samples at 12,000 x g for 15 min at room temperature. Forthe reaggregation experiments, 200 pL of GdmCI-solubilised IBs were diluted by dropwise addition of 5000 pL of MilliQ water. Expression kinetics of mCherryP4o IBs

[0348] All six p40 deletion variants (designated minus 1 to minus 6) were fused to mCherry, and their expression kinetics were assessed using a plate-based fluorescence assay under varying IPTG concentrations (0.1 , 0.4, and 1 mM) at 37 °C for 12 hours. Briefly, an overnight 5 mL seed culture was grown on LB broth supplemented with carbenicillin (100 pg / mL) and incubated at 37°C. 50 mL of LB broth was inoculated with 2% (v / v) of the seed culture and incubated at 37°C until the ODsoo reached 0.6-0.8 (mid-log phase). The protein expression kinetics was performed in a Greiner p clear® flat bottom (chimney well) 96-well microtiter plate (Greiner bio-one, Austria). 180 pL of mid-log phase culture was aseptically transferred into individual wells. The expression of the target protein was induced with 20 pL IPTG to obtain a final concentration between 0.1 and 1 mM. Samples without IPTG were also included as pre-induced control. Cultivation and real-time mCherry fluorescence measurements were performed simultaneously using a FLUOstar Omega multi-mode microplate reader (BMG Labtech, Germany). The cultivation and expression kinetic measurement parameters were as follows, incubation temperature at 37 °C with double orbital shaking at 200 rpm, and measurement mode were gain adjusted with the excitation and emission wavelengths of 595 and 630 nm, respectively. Direct measurement of mCherry fluorescence was recorded at the interval of 5 min for the total duration of induction. mCherry fused to p40 without deletions (full) was also assayed in the similar conditions. All the experiments were performed in three replicates and the results show the mean ± standard deviation (SD).

[0349] Cellular localisation of mCherryP4o deletions

[0350] Cells obtained from the above experiments were collected by centrifugation (4000 x g 3 min, 4 °C). The pellets obtained from 1 mL of expression culture were washed and resuspended with 200 pL of 1x PBS buffer to the final confluency at ODsoo of approximately 1 . Subsequently, 2 pL of the suspension was pipetted onto the agarose pad of a prepared microscope slide, covered with a glass cover slip. Imaging of mCherryP4o deletion variants IBs was performed using an Olympus FV3000RS confocal microscope (Olympus, Japan). The fluorescence of mCherryP4o deletion variants IBs was recorded with the inbuilt Ex (595 nm) / Em (630 nm) filters and all images were captured at a magnification of 100x.

[0351] Field Emission Scanning Electron Microscopy (FESEM) characterisation of p40 deletions

[0352] The morphology and size of all purified IBs formed by the p40 deletions fused to each of the four target proteins were investigated using field emission scanning electron microscopy (FESEM). The purified IBs were diluted 100-fold using ultrapure MilliQ and vortexed gently. 1 mL of diluted samples were then drawn using a sterile syringe and passed through a 0.22 pm polycarbonate isopore membrane (Merck, USA) mounted on a 13 mm Swinnex filter holder (Merk, USA). 500 pL of ultrapure water was passed through the membrane and excess water was displaced by passing air through the membrane using an empty syringe. The membrane was air-dried under a laminar airflow hood before transferring to the sample stub and fixed using carbon adhesive tape. The membrane was gold sputtered using Emitech K550 under argon atmosphere and images were captured at 5 kV of the acceleration voltage using a FESEM JSM- 71 OOF (JEOL, Japan). The size of IBs was measured by calculating the dimensions of ~50 IB structures using Imaged software (National Institutes of Health, USA). Functionalisation of different matrices

[0353] Different types of commercially available materials were studied for the functionalisation experiments. These are shown in Table 6.

[0354] Briefly, 200 pL of completely solubilised p40 IBs were added to the matrices and allowed to adsorb for 5 min. The reaggregation process was initiated by the dropwise addition of 5000 pL of MilliQ water under gentle agitation. The functionalised matrices with the reaggregated IBs was incubated in the MilliQ suspension for 5 min with gentle agitation, then it was transferred to a sterile petri dish and air dried to remove excess MilliQ.

[0355] Table 6 Matrices utilised with the p40 IBs

[0356] Functionalisation of CBSP097 matrix with enzymesP4o deletion variants

[0357] Material biofunctionalisation experiments were performed as described above. Standard enzyme assay (see below) was performed to determine the retained catalytic activity in the respective enzyme functionalised matrices.

[0358] Enzyme assays

[0359] P40 constructs p40 based a-amylase, -xylanase, a-glucan phosphorylase, glucose-6-phosphate isomerase, phosphoglycolate phosphatase, tagatose 4-epimerase and fructose 6-phosphate 4- epimerase activity in the form of both intact IBs and functionalised matrices were determined using the following standard enzyme assays. The standard reaction with the bare matrices alone incubated with the respective substrate was treated as blank. All the experiments were performed in triplicate, and the results are expressed as mean values. a-amylase activity was determined using potato starch as substrate. The standard assay reaction mixture contained 0.5% (w / v) potato starch, 50 mM HEPES buffer (pH 7.8) and enzyme functionalised matrices; the final volume was 200 pL. The reaction mixture was incubated at 80°C for 10 min.

[0360] P-xylanase activity was determined using oat spelt xylan as substrate. The standard assay reaction mixture contained 0.5% (w / v) oat spelt xylan, 50 mM sodium phosphate buffer (pH 6.5) and enzyme functionalised matrices; the final volume was 200 pL. The reaction mixture was incubated at 50°C for 10 min. The released free sugars after the enzyme reaction were determined by the DNS assay. a-glucan phosphorylase activity was determined using maltodextrin as substrate. The standard assay reaction mixture contained 1% (w / v) maltodextrin, 10 mM inorganic phosphate, 50 mM HEPES (pH 8) and enzyme functionalised matrices; the final volume was 200 pL. The reaction mixture was incubated at 70°C for 10 min. The conversion of maltodextrin into a- glucose-1-phosphate (a-G1 P) was determined using a discontinuous couple enzyme (CE) assay. Briefly, 100 pL of the reaction product was mixed with 2x CE mix containing 5 mM MgCl2, 1 mM NAD+, 2 U phosphoglucomutase (from rabbit muscle) and 1.4 U of glucose-6-phosphate dehydrogenase (from Leuconostoc mesenteroides) to the final volume of 200 pL where the formation of NADH was measured at A340 nm.

[0361] Phosphoglucomutase (PGM) activity was determined using glucose-1-phosphate (G1 P) as substrate. The standard assay reaction mixture contained 5 mM G1 P, 5 mM MgCl2, and 50 pM glucose 1 ,6 bisphosphate in 50 mM HEPES (pH 8) and enzyme functionalised matrices; the final volume was 100 pL. The reaction mixture was incubated at 70°C for 10 min. The conversion of glucose-1- phosphate (G1 P) into glucose-6-phosphate (G6P) was determined using a discontinuous CE assay. Briefly, 50 pL of PGM reaction product was mixed with 2x CE mix containing 5 mM MgCl2, 1 mM NAD+, and 1 .4 U of glucose-6-phosphate dehydrogenase in 100 mM HEPES (pH 8) to the final volume of 100 pL where the formation of NADH was measured at A340 nm.

[0362] Phosphoglucoisomerase (PGI) activity was determined using fructose-6-phosphate (F6P) as substrate. The standard assay reaction mixture contained 5 mM F6P, 5 mM MgCl2, 50 mM HEPES (pH 8) and enzyme functionalised matrices; the final volume was 100 pL. The reaction mixture was incubated at 70°C for 10 min. The conversion of F6P into G6P was determined using a discontinuous CE assay. Briefly, 50 pL of PGI reaction product was mixed with 2x CE mix containing 5 mM MgCl2, 1 mM NAD+, and 1.4 U of glucose-6-phosphate dehydrogenase in 100 mM HEPES buffer, pH 8 to the final volume of 100 pL where the formation of NADH was measured at A340 nm.

[0363] Phosphoglycolatephosphatase (PGP) activity was determined using the substrate analogue p-nitrophenyl phosphate (p-NPP). Briefly, the standard assay reaction mixture contained 10 mM p-NPP, and 5 mM MgCI2 in 50 mM HEPES (pH 8) and enzyme functionalised matrices; the final volume was 100 pL where the conversion of non-chromogenic p-NPP substrate into chromogenic p-nitrophenyl compound by PGP was measured at A410 nm. The standard reaction with the matrices functionalised with p40 alone incubated with the respective substrate was treated as blank.

[0364] Fructose 6-phosphate 4-epimerase (F6PE) activity was determined using fructose-6- phosphate (F6P) as the substrate. The standard reaction mixture (final volume: 200 pL) contained 5 mM F6P, 5 mM MgCI2, 50 mM phosphate buffer (pH 8.0), and enzyme-functionalised matrices. The reaction was incubated at 70 °C for 10 min. The conversion of F6P to tagatose-6- phosphate (T6P), and the subsequent dephosphorylation of T6P to D-tagatose, was monitored using a discontinuous capillary enzyme (CE) assay. Specifically, 100 pL of the F6PE reaction product was mixed with 2 mg / mL of PGPP4o and incubated at 70 °C for 10 min. The final product was diluted 50-fold and analysed by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). Analytical separation was performed using a Dionex ICS-6000 system equipped with a CarboPac PA-200 analytical column (3 x 250 mm) and a CarboPac PA-200 guard column (3 x 50 mm). Elution was carried out using a gradient of water and 200 mM NaOH with the following profile (v / v): 92:8 to 20:80 over 0-26 min, 20:80 to 0:100 from 26-29 min, and re-equilibration from 0:100 to 92:8 over 29-32 min.

[0365] Tagatose 4-epimerase (T4E) activity was assayed using D-fructose as the substrate. The standard reaction mixture (final volume: 200 pL) consisted of 5 g / L D-fructose, 1 mM Ni2+, 50 mM Tris-HCI buffer (pH 8.5), and enzyme-functionalised matrices. The reaction was incubated at 80 °C for 15 min. To quantify the conversion of D-fructose to D-tagatose, the reaction product was diluted 100-fold and analysed using the same Dionex ICS-6000 system and chromatographic conditions described for the F6PE assay.

[0366] Enzyme assay of P40 deletion mutants

[0367] For the p40 deletion mutants, a-amylase activity was determined using potato starch as substrate. The standard assay reaction mixture contained 0.5% (w / v) potato starch, 50 mM HEPES buffer (pH 7.8) and respective deletion variants of BactAmyP4o; the final volume was 200 pL. The reaction mixture was incubated at 80°C for 10 min. Enzymatic conversion was quantified using the dinitrosalicylic acid (DNS) colorimetric assay, by measuring reducing sugars released at 540 nm. The substrate-only reaction without BactAmyP4o served as the blank control. a-glucan phosphorylase activity was determined using maltodextrin as substrate. The standard assay reaction mixture contained 5% (w / v) maltodextrin, 10 mM inorganic phosphate, 50 mM HEPES (pH 8) and respective aGPP4o_full, minus 3 and minus 4 IBs; the final volume was 200 pL. The reaction mixture was incubated at 70°C for 10 min with stirring at 800 rpm.

[0368] Phosphoglycolatephosphatase (PGP) activity was determined using the substrate analog p-nitrophenyl phosphate (p-NPP). Briefly, the standard assay reaction mixture contained 10 mM p-NPP, and 5 mM MgCl2 in 50 mM HEPES (pH 8) and PGPP4o_full, minus 3 and minus 4; the final volume was 200 pL where the conversion of non-chromogenic p-NPP substrate into chromogenic p-nitrophenyl compound by PGP was measured at absorbance (A410 nm). The standard reaction with bare p40 alone incubated with the respective substrate was treated as blank.

[0369] Protein content estimation of o40 based IBs

[0370] The molar extinction co-efficient and molecular weight of all p40-based IBs were calculated from theirdeduced amino acid sequences using Expasy ProtParam tool. 3 mg of each p40-based IBs were taken and resuspended to 300 pL using solubilisation buffer containing 5 mM DTT in 50 mM Tris-HCI, pH 8.5. The suspensions were then vigorously pipetted and vortexed until no visible clumps are observed. 100 pL of homogenous suspension containing 1 mg of IBs was transferred into 3 different reaction tubes to facilitate triplicate measurement. The samples were further solubilised by adding solubilisation buffer containing 3 M GdmCI leading to a final concentration of 1 mg / mL IBs for each tube. After incubation at room temperature for 1 h with gentle agitation, the samples were subsequently centrifuged for 20 min at 12000 rpm to separate the soluble protein from any remaining insoluble debris. The absorbance (A280 nm) of each solubilised IBs solution was directly measured using NanoDrop One / Onec(Thermo Fisher Scientific) instrument. The A280 absorbance of each sample was obtained after blanking the A280 for solubilisation buffer containing 3 M Gdmcl with the baseline correction at 350 nm. The protein concentration in 1 mg / mL of each IBs solution was determined using previously calculated theoretical molar extinction coefficient by applying the Beer-Lambert Law formula where eProtein is concentration of target protein (M), A280nm- is the absorbance at 280nm, EProtein- is the theoretical molar extinction coefficient of the target protein (calculated using Protparam tool) and I- is the pathlength (mm).

[0371] Finally, the protein content within each IBs was calculated using the following equation:

[0372] All the experiments were performed in triplicate, and the results are expressed as mean values.

[0373] The protein content of the p40 deletion variants were also determined as per the preceding paragraphs.

[0374] SDS-PAGE analysis

[0375] Sodium dodecyl sulphate-polyacrylamide gel electrophoreses (SDS-PAGE) was used to visualise all p40-based IBs. Proteins were separated by electrophoresis using the Bio-Rad Mini- Protean TGX system (Bio-Rad Laboratories, USA). Samples were mixed with equal volumes of 2X Laemmli loading sample buffer and incubated at 95°C for 5 min. Denatured proteins were briefly centrifuged and loaded into pre-cast Bio-Rad Mini-PROTEAN® TGX gels (4-20%) and run at 100 V for 60 min in running buffer (25 mM Tris, 192 mM glycine, 1 % (w / v) SDS, pH 8.3). The protein bands were visualised by staining with Coomassie Brilliant Blue G-250 safe stain followed by destaining with MilliQ water. Gel images of the resolved protein bands were obtained using the G:Box F3 Gel Doc system (Syngene, UK). The Precision Plus Protein Dual Colour Standard (Bio-Rad) was included in all gels. Functionalisation efficiency

[0376] Using the estimated protein content of p40-based IBs, the efficiency of the functionalisation approach was assessed. The amount of protein in intact IBs and residual aggregate after functionalisation was determined by solubilising the pellet in 200 pL solubilisation buffer containing GdmCI (between 2-3 M) and 5 mM DTT in 50 mM Tris-HCI, pH 8.5. After 2 h of solubilisation, the solubilised mix was centrifuged shortly at 12000 rpm to remove nonsolubilised debris. The protein content was directly quantified using a Nanodrop One UV-Vis spectrophotometer. The obtained A280 absorbance was used to calculate the target protein concentrations by Beer-Lambert law (as per above formula).

[0377] Functionalisation efficiency was calculated using the following expression. where % entrapped is the concentration of target protein after functionalisation, %A280nm (SM)- is the concentration of target protein in starting material (SM) / intact IBs and %A280nm (residual IBs)- is the concentration of target protein in free aggregates (after functionalisation). All the experiments were performed in triplicate, and the results are expressed as mean values.

[0378] Functionalisation characterisation

[0379] Confocal laser scanning microscopy (CLSM)

[0380] The bright fluorescence of mCherry and eGFP protein were tracked using CLSM, that aided in understanding the changes in surface texture of all the matrix after functionalisation. The matrices treated with soluble mCherry and eGFP were considered as the functionalisation control. To visualise under CLSM, the matrices functionalised with either mCherryP4o or eGFPP4o were removed from the MilliQ suspension and allowed to air dry at room temperature. The airdried matrices were transferred into a black 24 well plate with flat and clear bottom. The fluorescence from control and matrices functionalised with mCherryP4o and eGFPP4o IBs was recorded with the inbuilt filters of Ex (595 nm) / Em (630 nm) and Ex (488 nm) / Em (510 nm), respectively. To visualise the functionalisation with enzymeP4o IBs, the matrices were stained using a 25-fold diluted SYPRO™ Orange Protein Gel Stain (5000 X, Thermo Fisher Scientific). The fluorescence was observed at Ex (490 nm) / Em (585 nm). All images were captured at a magnification of 10x. Field emission-scanning electron microscopy (FESEM)

[0381] The functionalised nonwoven fabric matrices were air-dried to remove excess MilliQ and mounted on 12.5 mm (diameter) sample stub using adhesive carbon tape. Functionalised spherical matrices like BioCradle, Cytodex and Immobeads 150P were drawn using a sterile syringe and passed through a 0.22 pm polycarbonate isopore membrane (Merck, USA) mounted on a 13 mm Swinnex filter holder (Merk, USA). The excess water was displaced by passing air through the membrane using an empty syringe. The membrane was air-dried under a laminar airflow hood before transferring to the sample stub and fixed using carbon adhesive tape. All samples were gold sputtered using Emitech K550 under Argon atmosphere and images were captured at 5 kV of the acceleration voltage using a field emission scanning electron microscope (FE-SEM) JSM-7100F (JEOL, Japan).

[0382] Attenuated total reflection-Fourier emission-scanning infrared (ATR-FTIR) spectroscopy

[0383] Before collecting ATR-FTIR spectra, all the functionalised CBSP097 matrices were airdried at room temperature overnight for the maximal removal of adsorbed water. The FTIR spectra of bare and functionalised matrices (mCherryP4o and BactAmyP4o) were recorded on a JASCO FTIR-4700 spectrometer (JASCO, Japan) attached to single reflection ATR module consisted of monolithic diamond crystal platform with a triglycine sulfate (TGS) detector at a resolution of 4 cm-1 and 64 accumulated scans in the wavenumber range of 4000-400 cm-1. Three replicate spectra were averaged for every sample. A background scan was performed immediately prior to each sample measurement.

[0384] Recyclability

[0385] Standard enzyme assay was followed to determine the recyclability / operational stability of enzyme functionalised matrices. The recyclability of the enzyme functionalised matrices was determined by running the assays over 10 cycles. Briefly, each enzyme functionalised matrices (fibres size were 2 cm in diameter and beads / particles were 30 mg) were taken in a 1 .5 mL microcentrifuge tube, mix with their respective substrate (200 pL) and incubated at their respective reaction temperature. The reaction was stopped by placing tubes on ice-bath for 5 min. The matrices with reaction mix were centrifuged shortly before each cycle and the collected reaction mix was assayed for DNS or CE assay as described in standard enzyme assay. Fresh substrate was added after each cycle. The relative activity (%) of recycling was calculated using the following equation,

[0386] Operational stability (%) = [An / Ai] x 100 where An is the enzyme activity after each cycle and Ai is the initial enzyme activity. Effect of solubilising agents on solubilisation and reaqqreqation of intact p40 IBs

[0387] To assess the solubilisation and reaggregation capabilities of p40-based IBs while retaining functionality, various solubilisation methods were investigated. These methods included mild denaturing chaotropes such as guanidinium chloride (GdmCI), guanidine thiocyanate (GdSCN), urea, and L-arginine. Additionally, anionic surfactants like sodium dodecyl sulfate (SDS) and N-lauroylsarcosine sodium salt (NLS) were prepared in a solubilisation buffer containing 50 mM Tris-HCI (pH 8.5) and 5 mM DTT. Low pH solubilisation (pH 4-6) utilised a 2 M sodium acetate bufferwith 400 pM MgCl2 and 5 mM DTT, while high pH (pH 8-12) solubilisation was carried out using 2 M Tris-HCI buffer with 5 mM DTT. Approximately 4 mg of mCherryP4o was solubilised separately with 200 pL of solubilising buffer containing either 3 M GdmCI, 3 M GdSCN, 3 M urea or 1 .5 M L-arginine. Samples solubilised for 2 h at room temperature with gentle agitation. Similarly, for surfactants, 200 pL of either2% SDS or O.5% NLS in 2 M respective buffer solutions were used for solubilising.

[0388] Solubilisation was confirmed by the absence of a visible pellet after centrifugation of the samples at 12,000 x g for 15 min at room temperature. The turbidity measurement at A350 nm also confirmed the complete solubilisation. For the reaggregation experiments, 200 pL of solubilised mCherryP4o from each condition were diluted by dropwise addition of 5000 pL MilliQ water. All experiments were carried out in three independent replicates.

[0389] Bifunctionalisation of polypropylene (PP) fibre

[0390] Equal amounts of BactXylBP4o and BactAmyp40 IBs pellets (~8 mg each) were solubilised using 200 pL of 3 M Guanidine chloride (GdmCI) for each IBs. A circular-shaped PP fibre was cut and the 200 pL of solubilised BactXylBP4o was added onto one half of the fibre which was then allowed to absorb for 5 min. Similarly solubilised BactAmyp4o was added to the other half of the circular-shaped fibre. Bifunctionalisation of PP fibre with both the IBs was started by dropwise addition of MilliQ (up to 6 mL). The fibre with reaggregated IBs was allowed to resuspend in MilliQ for another 5 min. Subsequently, the functionalised fibre was allowed to air-dry at RT and then transferred into a sterile 10 mL syringe without plunger. The syringe nozzle was sealed using silicone end cap and 400 pL of 0.5% xylan substrate solution was added onto the biofunctionalised PP fibre inside the syringe and incubated at 50°C for 15 min. The xylanase reaction was stopped by placing the syringe in an ice bath. The reaction mixture trapped within the fibre was dispensed using the plunger and approximately 400 pL of reaction mixture was extracted. Following that, 400 pL of 0.5% starch substrate was introduce into the syringe to test BactAmyP4o activity at 80°C for 15 min. This was repeated up to 5 cycles for each enzyme reaction. At the end of each cycle, the reducing sugars in each extract were determined using the DNS assay. Recyclability of PP fibre-functionalised T4EP4o using SpinChem® rotating bed reactor (RBR)

[0391] Enzymatic reactions were performed using the SpinChem® RBR S2 system (SpinChem AB, Sweden) to evaluate the feasibility of employing enzyme-functionalised matrices at a small pilot scale. The RBR S2 basket, comprising four compartments, was loaded with T4EP4o- functionalised polypropylene (PP) fibre at an approximate concentration of 20 mg / mL (wet weight basis) per compartment. The jacketed reaction vessel was preheated to 80 °C prior to substrate addition. The reaction mixture consisted of 120 mL of substrate solution containing 5 g / L D- fructose and 1 mM Ni2+in 50 mM Tris-HCI buffer (pH 8.5). After equilibrating the solution at 80 °C for 5 min, the loaded RBR basket was submerged into the reaction vessel. Each reaction cycle was run for 120 min with constant stirring at 800 rpm. At the end of each cycle, the reaction mixture was drained, and any residual product entrapped within the RBR basket was recovered by briefly increasing the stirring speed before draining. Successive cycles were initiated by adding 120 mL of fresh substrate solution to the vessel. Samples were collected at the end of each cycle for product analysis by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

[0392] Multi-enzyme conversion of maltodextrin to D-taqatose using enzyme-functionalised matrices

[0393] A modular five-enzyme pathway was employed to convert maltodextrin to the rare sugar D-tagatose. The pathway comprised a-glucan phosphorylase (aGP), phosphoglucomutase (PGM), phosphoglucoisomerase (PGI), fructose-6-phosphate 4-epimerase (F6PE), and phosphoglycolate phosphatase (PGP). Each enzyme was expressed as an insoluble p40-fusion (enzymeP4o) and subsequently functionalised onto CBSP097 matrices at a concentration of 20 mg / mL (wet weight basis). The kinetic parameters of all five enzymes are summarised in Table 7.

[0394] Table 7 Kinetic parameters of enzymesP4o used in multi-enzyme pathway

[0395] Specific Km Vmax (mM Kcat Kcat / Krn(mM-1

[0396] EnzymeP4o Substrate activity

[0397] (mM) min-1) (min-1) min-1)

[0398] (U / g) aGPP4o maltodextrin 0.58 0.43 358.33 12.05 20.77

[0399] PGMP4O G-1-P 0.10 0.06 50.00 0.73 7.31

[0400] PGiP4o G-6-P 16.47 1.28 1066.67 36.25 2.20

[0401] GatZP4o F-6-P 7.71 0.07 58.33 1.37 0.18

[0402] PGPP4O D-fructose 10.49 0.24 200.00 3.16 0.30 Prior to the reaction, the functionalised matrices were air-dried and equilibrated in phosphate buffer (50 mM PB, pH 8.0). The initial reaction mixture, composed of 10 g / L maltodextrin, 10 mM inorganic phosphate, and 5 mM MgCI2in 50 mM PB (pH 8.0), was added to a tube containing CBSP097-functionalised aGPP4o and incubated at 70 °C for 60 minutes with constant stirring at 800 rpm. Sequential conversions were performed by transferring the reaction mixture through matrices functionalised with the remaining enzymes (PGMP4o- PGiP4o- F6PEP4O- PGPP4O), under identical reaction conditions. Samples were collected at each enzymatic step and analysed for product titres using HPAEC-PAD. A control reaction using free enzymep4o preparations was conducted in parallel under the same conditions to assess the impact of immobilisation on overall conversion efficiency.

[0403] Production of Functional / catalvticallv active IBs using o40 fusion

[0404] The expression plasmids carrying different proteins fused to p40 were introduced into E. coli Tuner® (DE3) cells and recombinant protein production and purification of p40 based IBs were carried out as described in Materials and Methods. After purification, the purity and yield of different p40 IBs was evaluated by SDS-PAGE analysis (Figure. 1A). Despite similar culture / growth conditions, observations from SDS-PAGE showed that the IBs were produced with different yields based on protein band intensities. This observation was consistent with the protein content estimation results (Figure. 1 B) where aGPP4o retained highest protein content, while other IBs showed comparable protein concentration.

[0405] The characteristics ofthe different p40 based IBs are shown in Table 8.

[0406]

[0407] Characterisation of functionalised matrices

[0408] The intrinsic ability of solubilised p40 based IBs to reaggregate upon diluting the chaotropic (solubilising) agent formed the framework of our material functionalisation strategy. The nature of functionalisation and surface texture of different matrices before and after functionalisation was studied using Confocal laser scanning microscopy (CLSM) and field emission scanning electron microscopy (FESEM). Both microscopy techniques were able to distinguish the characteristics of functionalisation on different support matrices.

[0409] Evaluation of storage stability of BactAmyP4o functional matrix

[0410] The shelf life of a-amylase (BacAmyP4o) in functionalised CBSP097 matrices during storage were assessed at both 4 and 25°C. On average each matrix was functionalised with 2.5 mg / mL BactAmyP4o. The stability was determined by measuring the amylase activity of functionalised matrices weekly for a period of 8 weeks. The relative weekly activities were compared with the initial activity measured during Oth week. For each week and for both temperatures, matrices were functionalised independently. Each result was calculated by averaging two replicate experiments.

[0411] Solubilisation and reaggreaation of D40-homologs and matrix functionalisation

[0412] To test the potential of other members of Auxiliary Activity Family 10 (AA10) to form IBs, three p40-homologs from different origin namely, Kibdelosporangium aridum (KaridP4o), Archangium lipolyticum (AlipoP4o), and Phytohabitans suffuscus (PsuffP4o) were recombinantly produced as IBs in E. coli. The IBs obtained were purified as described for p40-based IBs. All three new p40-homologs were tested for their intrinsic solubility-reaggregation property. Therefore, solubilisation and reaggregation experiments were carried out as described for p40. Briefly, ~8 mg of purified IBs was solubilised using 200 pL of 3 M GdmCI for 1-2 h at room temperature. This was followed by centrifugation to remove the any remaining particulates and other debris. Then the clear supernatant was added to a circular shaped CBSP097 matrix and allowed to adsorb for 5 min. The reaggregation process was initiated by the dropwise addition of 5000 pL MilliQ water under gentle agitation. The functionalised matrices with the reaggregated p40-homolog IBs was further incubated in the MilliQ suspension for 5 min with gentle agitation, then it was transferred to a sterile petri dish and air dried to remove excess water. The presence of aggregates in the functionalised matrices were confirmed using microscopic analysis after staining with fluorescent SYPRO Orange dye as described for p40-based IBs. Example 1 Functionalisation of synthetic nonwoven PP fibres with bioactive protein

[0413] Nonwoven polypropylene (PP) fabrics find their application in various fields because of their excellent mechanical properties such as high heat and moisture retention, light weight, porous, and superior moisture- vapour permeability. These qualities of PP fibre made it an appealing candidate for enzyme immobilisation, particularly the high porosity and permeability nature can enable high mass transfer during enzyme reactions in a membrane bioreactor. To demonstrate the applicability of the inventors functionalisation strategy, synthetic nonwoven PP fibres were functionalised with 4 different p40-based IBs namely eGFPP4o, mCherryP4o, BactXylBP4o and BactAmyP4o. In all cases, the reaggregated IBs were observed to be trapped within the fibre mesh. The presence of distinct fluorescence spots within the fibre monomers indicates that the aggregates of eGFPP4o and mCherryP4o are not just physically adsorbed but compactly bound over the individual fibres (Figure. 2B and 2D). The controls samples with fibre treated with the soluble form of eGFP (Figure. 2A) and mCherry (Figure. 2C) showed negligible fluorescence which implied that soluble proteins were not capable to functionalise the fibre material . Although the nature of functionalisation was found to be uniform irrespective of p40- based IBs, the fibre functionalised with BactAmyP4o showed aggregates of larger size when compared to fluorescent p40 IBs (see Figure. 2B).

[0414] The Field Emission Scanning Electron Microscopy (FESEM) images (Figure 3) of functionalised PP fibre showed a distinguishable difference in surface texture from bare fibres. The mCherryP4o-functionalised fibre monomer appeared to possess reaggregated p40 IBs, mostly in the form of glued over the fibre filaments thereby attaching the filaments together (Figure. 3B). A few regions of functionalised fibre showed that the aggregates were hanging from the surface. However, the surface of fibre filament that treated with mCherry protein without p40 (Figure. 3A) retained theirsmoothness whereas the functionalised filament showed rough texture bounded with aggregates. This confirmed that the solubilised p40 IBs added to the PP fibre can be reaggregate upon removal of chaotropic agents.

[0415] Example 2 Functionalisation of cellulose microfibers (CBSP060, CBSP100) with bioactive protein

[0416] Two types of commercially available Microline cellulose-based nonwoven fabrics, which are used as sample pads for in vitro diagnostics (IVD), were tested. Both CBSP060 and CBSP100 are continuous filament hydrophilic fabrics without binders, with very little lint and thicknesses of 0.47 and 0.39 mm, respectively. The functionalised fabrics showed the aggregated mCherryP4o IBs adhering all overthe surface ofthe fibre filament (Figure. 4B and 5B). CBSP060 contained fluorescent aggregates that dispersed throughout the fibre mesh (Figure. 4B) while CBSP100 showed uniformly fluorescent filaments (Figure. 5B). In both cases, the fibre treated with the soluble mCherry (without p40) did not show any bright fluorescence indicating that the fibres alone cannot take up any solubilised protein (Figure. 4A and 5A).

[0417] Despite their hydrophilic cellulosic fibre, CBSP060 and CBSP100 showed differences in the pattern of functionalisation under FESEM (Figure. 6A and 7A). The formation of aggregates during functionalisation in CBSP060 appeared to bind along with the fibre filaments (Figure. 6B) whereas functionalised CBSP100 displayed distinct overhanging aggregates (Figure. 7B) and a few parts of fibre showed a thin film of aggregates that glued nearby filaments together. This difference in the functionalisation pattern can be attributed to the average filament size and spatial arrangement of CBSP060 and CBSP100 fibres, where CBSP060 contains fibre filaments of size 14.9 pm while CBSP100 is 11.9 pm. Moreover, CBSP060 is a perforated fabric which may also influence in the formation of aggregation pattern during functionalisation.

[0418] Due to their excellent moisture absorption and desorption ability, the Microline range of microfibres were also tested with BactAmyP4o, the results are presented in the following examples.

[0419] Example 3 Functionalisation of cellulose / PET microfibres (CBSP097) with bioactive protein

[0420] A further in vitro diagnostic lateral flow material investigated was CBSP097 which is also a continuous filament nonwoven and highly uniform hydrophobic fabric comprising cellulose and PET microfibres arranged as a wave pattern with a thickness of 0.34 mm and an average individual fibre filament size is 11.2 pm. CLSM images of mCherryP4o-functionalised CBSP097 (Figure. 8) showed a similar appearance to that of CBSP060 and CBSP100. After functionalisation, scattered fluorescence of mCherryP4o was observed along the length ofthe fibre filaments (Figure. 8B) which was not seen in fibres treated with soluble mCherry (Figure. 8A). Further, investigation by FESEM showed the presence of CBSP097 embossed pattern of aggregation with a highly rough surface texture (Figure. 9B). The adjacent fibre filaments in the functionalised matrix were found to be stuck together. This pattern was absent in the bare matrix treated with soluble mCherry (Figure. 9A) which matrix retained their smooth surface.

[0421] Example 4 Functionalisation of PET microfibers (PMAP080, PMAP090 and PSCP250) with bioactive protein

[0422] PET microfibers are known for their attractive mechanical properties including high resistance, elasticity, and rapid drying. They are highly uniform ultra-fine continuous filament nonwoven fabrics. Due to their excellent liquid adsorption and impurity separation quality, PMAP080, PMAP090 and PSCP250 are used as adsorbent pads for IVD. The confined red fluorescence throughout the surface of microfibrils confirmed the functionalisation ofthe matrices with mCherryP4o (Figure 10B and 12B). Samples treated with soluble mCherry displayed no red fluorescence (Figure. 10A and 12A). While mCherryP4o-functionalised PMAP080 and PMAP090 seemed comparable in CLSM images, FESEM analysis revealed a significant difference in their functionalisation pattern (Figure. 11A and 13A). With PMAP080 the reaggregated mCherryP4o appeared as slightly granulardeposits that were embedded overthe fibre’s surface, a few regions showed amalgamation of adjacent fibrils due to the reaggregated IBs (Figure. 11 B). On the other hand, PMAP090 showed flatten, overhanging mCherryP4o aggregates that firmly bind the fibrils together (Figure. 13B).

[0423] In contrast to PMAP080 and PMAP090, mCherryP4o-functionalised PSCP250 showed isolated patches of aggregates over fluorescent filaments (Figure. 14B). Under a FESEM, there were noticeable differences in the surface texture between bare and functionalised PSCP250 (Figure. 15).

[0424] All 3 samples treated with soluble mCherry presented highly smooth and uniform morphology (Figure. 11 A, 13A and 15A) while those functionalised with mCherryP4o showed rough, compressed, and irregular structure with coarse surface patches indicating the strong binding of aggregates as result of functionalisation (Figure.11 B, 13B and 15B). Regardless of the material class, these microfibres exhibit noticeable variations in the appearance of functionalised fibrils. This can be explained by the variation in the average size and thickness of the fibres (monomers). Among 3 PET fabrics, PSCP250 possess densely arranged fibrils with size of 21.6 pm and thickness of 0.54 mm while monomers of PMAP080 and PMAP090 fabric are less dense with size and thickness of 2.9 and 1.7 pm and 0.19 and 0.38 mm, respectively. Thus, the mechanisms by which functionalisation occur might be determined by the size and thickness of material.

[0425] Example 5 Functionalisation of macroporous cellulose beads with bioactive protein

[0426] Microcarrier mediated cell culturing are a widely employed cultivation technique in the commercial production of biologies, vaccines, and biopharmaceuticals. Here, the microcarriers function as an excellent support facilitating larger surface area for enhanced cell adherence and proliferation. BioCradle L is a new class of microcarrier composed of macroporous cellulose beads with positively charged N, N-diethylaminoethyl (DEAE) surface groups (low charge). As a cellulosic material, the macroporous BioCradle offers excellent cell adherence and mechanical stability under agitation, promoting increased cell density and higher product yield. BioCradle was selected as a tested matrix for our application based on its unique characteristics, which include mechanically stability, large surface area with particle size 200-280 pm, and macroporous nature with average pore diameter of 30 pM. mCherryp4o-functionalised BioCradle showed the packing of fluorescent aggregates from surface to interior (Figure. 16B). Conversely, BioCradle treated with soluble mCherry was not fluorescent which implies that bare BioCradle beads are not capable of taking up any soluble protein (Figure. 16A).

[0427] Morphological changes in the functionalised beads were observed using FESEM imaging which showed beads decorated with mCherryP4o aggregates around the surface and within the pores (Figure. 17B). Biocradle beads treated with soluble mCherry retain their original structure and intact pores (Figure. 17A).

[0428] Example 6 Functionalisation of dextran microcarriers with bioactive protein

[0429] Cytodexl is a cross-linked dextran based microcarrier generally used to culture anchorage-dependent cell lines. These microporous beads are biologically inert, non-rigid, transparent, and uniform in size (180 pm). They carry positively charged groups (DEAE) throughout the matrix which enable higher cell density. CLSM imaging showed that after functionalisation with mCherryP4o, the beads appeared clumped together and encapsulated by the aggregates (Figure. 18B). These aggregates were found to attach around the surface of the beads in the form of fluorescent ring. As result of functionalisation, the smooth surface of beads was replaced by the aggregates with rough texture (Figure. 19B). Cytodexl beads treated with soluble mCherry displayed no fluorescence (Figure. 18A) and retained their original smooth surface (Figure. 19A). In general, Cytodexl beads were less stable to the mechanical stirring used during the functionalisation process resulting in some collapsed beads (data not shown).

[0430] Example 7 Functionalisation of PE frits with bioactive protein

[0431] Polyethylene (PE) made frits are commonly used as anti-clogging bed support in high- performance liquid chromatography (HPLC) and solid phase extraction columns. The PE frit tested in this study was made of battery vent polyethylene sheet with a thickness of 3.2 ± 0.10 mm and uniform pore size of 84 pm. PE frits were functionalised with mCherryP4o. As shown in CLSM images (Figure. 20), after functionalisation mCherryP4o aggregates were dispersed within the inner core of the PE frit (Figure. 20B). PE frits treated with soluble mCherry did not retain any visible fluorescence (Figure. 20A).

[0432] Example 8 Functionalisation of Immobeads with bioactive protein

[0433] Immobeads 150P are cross-linked methacrylate copolymers carrying oxirane functional groups. These methacrylate beads are characterised to possess 0.15-0.50 mm particle size and used as a carrier for industrial enzyme immobilisation. Immobeads were evaluated as another solid matrix for our functionalisation platform. CLSM images of mCherryP4o-functionalised Immobeads 150P appeared to have bright fluorescent rings along their entire surface (Figure. 21 B) while no fluorescence was observed in beads treated with soluble mCherry (Figure. 21 A).

[0434] Unlike, BioCradle and Cytodexl , the mCherryP4o-functionalised Immobeads 150P did not exhibit the presence of distinct mCherryP4o aggregates over their surface except for isolated patches (Figure. 17B and 19B). Similar to other matrices, functionalisation resulted in agglomeration of beads where the mCherryP4o aggregate formed at junction between beads (Figure. 22 B).

[0435] Example 9 Functionalisation of polystyrene Bio-beads with bioactive protein

[0436] Bio-beads are nonpolar, highly thermostable polystyrene beads within the size range of 300-1180 pm. Each Bio-bead consist of multiple crosslinked microspheres that confers higher surface area and make these beads excellent adsorbent for wide range of solvents and aqueous media. To test the applicability of solubilisation and reaggregation strategy on non-uniform polystyrene particles, Bio-beads were functionalised with mCherryP4o. CLSM images showed negligible fluorescence signal in Bio-beads treated with soluble mCherry (Fig. 23A) whereas mCherryP4o-functionalised Bio-beads showed distinct bright fluorescence indicating their surface functionalisation (Fig. 23B). However, CLSM also showed that the functionalisation was non- uniform where smaller beads appeared as bright fluorescent particles than the larger beads. This was also reflected in the functionalisation efficiency, with only 72% of starting material functionalised (Fig. 25). This suggests that this functionalisation technique require further optimisation to account for carriers with non-uniform particle size.

[0437] Example 10 Co-functionalisation of PP fibre

[0438] In an effort to evaluate the versatility of our functionalisation technique, PP fibres were simultaneously co-functionalised with two different enzymeP4o, namely BactXylBP4o and BactAmyP4o. The operational stability of fibres carrying the two enzymep40 was assessed for 5 consecutive cycles with BactXylBP4o activity assessed first followed by BactAmyP4o. At the end of 5 cycles BactAmyP4o and BactXylBP4o retained 74% and 55% of their initial relative activity, respectively (Figure. 24). The optimum temperature for the BactXylBP4o activity assay was 50°C. The relative activity of BactXylBP4o gradually decreased over time and without wishing to be bound by theory, this could be due to the higher temperature (80°C) used to perform the reaction with BactAmyP4o at each cycle. Regardless of this, the successful co-functionalisation of a single matrix with the current method can form the basis to explore one-pot multi-enzyme biocatalysis. Example 11 Functionalisation efficiency

[0439] The physical property of 12 selected matrices (Table 6) and their influence in protein loading capacity and functionalisation efficient was studied by estimating the amount of protein retained after functionalisation with mCherryP4o and BactAmyP4o. Overall, with mCherryP4o and BactAmyP4o high functionalisation efficiency (82-100%) was achieved with most matrices (Figure. 25 and Table 9). Only a slight reduction of functionalisation efficiency was observed with perforated fabric like CBSP060 (84%) and macroporous BioCradle beads (89%).

[0440] For the matrices treated with BactAmyP4o, the functionalisation efficiency was additionally estimated by assaying the amylase enzyme activity retained by the functionalised matrix. Most matrices retained high amylase activity after functionalisation (between 100-75% relative activity) when compared to the starting free BactAmyP4o (Figure 26). Although Bio-beads and BioCradle displayed almost 100% BactAmyP4o functionalisation efficiency based on protein concentration, they displayed 61 and 58% amylase activity, respectively (Figure. 26). Previous experiments showed that BioCradle were decorated with mCherryP4o aggregates around the surface and within the pores of the beads (Figure. 16B and 17B). The reduction in amylase activity after BioCradle functionalisation with BactAmyP4o might be due to the inability of the substrate to reach inside the pores of the macroporous beads. Similarly, the reduced amylase activity in BactAmyP4o-functionalised Bio-beads may be due to their porous structure which may possibly impede substrate accessibility inside the pores.

[0441]

[0442]

[0443] One of the major advantages of immobilised enzyme over their free form is the ability to reuse them without losing their catalytic activity. In industry, the operational cost of enzymebased reactions can be decreased in large part by reusing them. In the following examples, the inventors have demonstrated the reusability of different enzyme functionalised matrices.

[0444] Example 12 Functionalisation of PP fibre with BactAmyp40

[0445] The functionalisation of PP fibre with BactAmyP4o was confirmed using confocal laser scanning microscopy (CLSM) after staining with fluorescent SYPRO Orange dye, which specifically stained the protein aggregates of BactAmyP4o within the functionalised fibre filament (Figure. 27). These aggregates appeared as distinct bright spots distributed across the surface of the filaments (Figure. 27B). Additionally, the CLSM images revealed a noticeable difference in surface texture between the fibre before and after functionalisation, with the presence of aggregates imparting a rough surface to the functionalised fibre filaments. As a control for the functionalisation experiment, the bare fibre treated with SYPRO Orange dye did not exhibit any fluorescence (Figure. 27A).

[0446] Example 13 Recycling of BactAmyp4o-functionalised PP fibre

[0447] The a-amylase derived from the extremophilic bacterium Bacillus licheniformis exhibits remarkable thermostability, efficiently catalysing the hydrolysis of glycosidic linkages within starch molecules to yield dextrose. Due to its capability to operate optimally at elevated temperatures, typically ranging between 80-90°C, this a-amylase is utilised across diverse industries such as biofuel production, starch valorisation, textiles, and detergent manufacturing. The operational stability BactAmyp4o-functionalised synthetic PP fibre upon recycling was investigated for 12 cycles at both 70°C and 80°C (Figure. 28). At 70°C the amylase activity of the BactAmyP4o-functionalised PP fibre was fully stable displaying still 100% relative activity after 12 cycles. At 80°C, a 30% reduction in initial relative activity was observed after 8 cycles. Despite the higher assay temperature, after 12 cycles still 50% of the initial activity was observed in the BactAmyp4o-functionalised PP fibres.

[0448] Example 14 Recycling of BactAmyP4o-functionalised Biocradle beads

[0449] Due to their macroporous nature, Biocradle cellulose beads are a promising matrix for enzyme functionalisation, enhancing mass transfer efficiency. A reusability experiment was conducted at 80°C using Biocradle functionalised with BactAmyP4o, demonstrating that the functionalised Biocradle maintained over 75% of its initial activity after 8 repetitive cycles (Figure. 29). Furthermore, it retained 53% of its initial activity even after 10 cycles. Example 15 Recycling of BactXylBP4o-functionalised PP fibre

[0450] Endo-1 ,4- -D-xylanases are industrially important enzymes crucial for sequestering xylan- containing agricultural biomass, enabling the bioproduction of xylooligosaccharides. These compounds serve as valuable agronomical yield enhancers, plant growth regulators, taste enhancers with prebiotic and immunomodulatory effects, and constituents of livestock feed (Yan, B. et al., (2022). Carbohydrate Polymers, 292, 1 19641). BactXylBP4o-functionalised PP fibre was subjected to recyclability testing at 40°C and 50°C (Figure. 30). The results indicated a similar trend in reusability efficiency at both temperatures. After 8 cycles, over 30% of the initial activity was still detected. However, prolonged incubation led to a decline in xylanase activity levels, reaching approximately 50% after 10 cycles

[0451] Example 16 Recycling of CBSP097(cellulose / PET multilayer microfibers) functionalised with different thermostable enzymes

[0452] Thermostable aGPpw a-glucan phosphorylase (aGP), a member of the glycosyltransferase family, plays a critical role in carbohydrate metabolism in both prokaryotes and eukaryotes. aGP is particularly involved in the breakdown of storage polysaccharides such as starch, glycogen, and maltodextrin (Colpaert, M., et al., (2020). In Amino Acid Biosynthesis ~ Pathways, Regulation and Metabolic Engineering (pp. 177-210), Ubiparip, Z., et al., (2018). Applied Microbiology and Biotechnology, 102(19), 8187-8202). In recent years, there has been increasing attention on identifying thermostable variants of aGP due to their potential applications in various industries, particularly in the bioproduction of numerous sugar derivatives, including nucleotide sugars, glycosides, and rare sugars. The inventors incorporated thermostable aGP from Dictyoglomus turgidum into CBSP097, a nonwoven fabric composed of cellulose and PET microfibers. This aGP is a homotetramer (around 65 kDa each monomer) with a native molecular weight of approximately 261 kDa (Dai, Y., et al., (2021) Applied Biochemistry and Biotechnology, 193(11), 3719-3731). The recycling efficiency of aGPP4o, both in free and functionalised forms, was tested over 10 repetitive cycles (Figure. 31). In both forms, aGPP4o maintained 100% of its initial activity after 10 cycles at 70°C.

[0453] Thermostable PGMpw

[0454] Phosphoglucomutase (PGM) is a versatile enzyme that plays a pivotal role in various metabolic pathways. PGM catalyses the bi-directional interconversion of a-D-Glucose-1- phosphate and a-D-glucose 6-phosphate (G6P), a key regulatory reaction in carbohydrate metabolism. Its function extend beyond carbohydrate metabolism, impacting specialised processes in both plants and animals. The inventors used the monomeric thermostable PGM (50.3 kDa) from Thermus thermophilus to functionalise CBSP097. The PGMP4o-functionalised CBSP097 was subjected to 10 cycles of reuse at 70°C (Figure. 32). Throughout the recycling process, the PGMP4o- functionalised CBSP097 exhibited increased stability, with relative enzyme activity between 125% and 140% of its initial activity afterthe first cycle. Similarly, the free PGMP4o retained 100% of its initial relative activity at the end of 10 cycles.

[0455] Thermostable PgiP4o

[0456] The applicability of the functionalisation strategy was tested with another regulatory enzyme, phosphoglucoisomerase (PGI), also known as glucose-6-phosphate isomerase. PGI plays a crucial role in glycolysis and gluconeogenesis processes by catalysing the interconversion of aldohexose into ketohexose, specifically converting glucose-6-phosphate to fructose-6- phosphate. The products from PGI reaction are a key intermediate in the pathway producing glucose, glycosides, and N-acetylglucosamine (Kim, S.-J., et al., (2020). Journal of Microbiology, 58(9), 725-733). In this study, CBSP097 was functionalised with the thermostable PGIP4O from Thermus thermophilus. The thermostable PGI from T. thermophilus is a homodimer (around 46 kDa each monomer) with a native molecular weight of approximately 92 kDa (Fujisawa, T., et al., (2017). Applied and Environmental Microbiology, 83(16), e00550-17, Wang, W. et al., (2017). Metabolic Engineering, 42, 168-174). Both, free PGIP4o and PGIP4o- functionalised CBSP097, remained stable during the 10 cycles test at 70°C (Figure. 33). PGIP4o- functionalised CBSP097 showed a slightly increased enzyme activity (between 110% and 130% of its initial activity).

[0457] Thermostable PGPpw

[0458] Phosphoglycolate phosphatase (PGP), also referred to as glyceraldehyde-3- phosphatase, is an enzyme involved in the metabolism of sugars, particularly in glycolysis and related pathways. Its primary function is the dephosphorylation of phosphoglycolate to glycolate, or glyceraldehyde-3-phosphate to glyceraldehyde, depending on the specific substrate.

[0459] The thermostable PGP from the archeon Archaeoglobus profundus is a homodimer (around 24 kDa each monomer) with a native molecular weight of approximately 48 kDa (Dai, Y. et al., (2022) Biochemical Engineering Journal, 178, 108303, Dai, Y. et al. ,(2020) Enzyme and Microbial Technology, 139, 109594). Both, free PGPP4o and PGPP4o-functionalised CBSP097 displayed high stability during the 10 cycles assay at 70°C (Figure. 34). The free PGPP4o exhibited a slightly increased enzyme activity (between 110% and 120% of its initial activity). The increase in stability was more remarkable with PGPP4o-functionalised CBSP097 showing values between 120% and 168% of its initial activity. Thermostable F6PEP4o

[0460] Enzymes that catalyse C4-epimerisation reactions are essential in the industrial synthesis of sugar derivatives. These biocatalysts enable stereochemical inversion at the C4 position of carbohydrate molecules, generating structurally distinct compounds with wide-ranging applications in pharmaceuticals, food technology, and biotechnology (Frey, P. A. (1996) FASEB Journal, 10, 461-470). For instance, UDP-glucose 4-epimerase (GalE) catalyses the reversible conversion of UDP-glucose to UDP-galactose, a key precursor in the biosynthesis of glycosylated compounds used in therapeutic and nutritional formulations (Chen, L. L. et al. ,(2018) Biochemistry (Moscow), 83, 37-44). Recent studies have identified a novel fructose- 6-phosphate 4-epimerase (F6PE) enzyme exhibiting high thermostability, which has been incorporated into multi-enzyme cascades for D-tagatose production from maltodextrin (Dai, Y. et al. ,(2020) Enzyme and Microbial Technology, 139, 109594). In this study, the thermophilic F6PEP4o from Caldithrix abyssi was immobilised on CBSP097 matrices. The F6PEP4o enzyme retained 87% of its initial activity after 10 repeated cycles, whereas the CBSP097-functionalised F6PEP4o retained 96% of its initial activity under identical conditions (Figure 41). This demonstrates a marked enhancement in operational stability upon immobilisation. The improved performance of the immobilised enzyme highlights the compatibility of the F6PEP4o-CBSPO97 system with high-temperature, repeated-use biocatalytic applications, supporting its potential use in industrial-scale processes involving thermally intensive reaction conditions.

[0461] Thermostable T4EP4o

[0462] A recently identified epimerase, tagatose 4-epimerase (T4E), was developed through structure-guided engineering oftagaturonate 3-epimerase, using structure- and sequence-based protein clustering strategies (Shin, K.-C. et al., (2020) ACS Catalysis, 10, 12212-12222) (Chen, J. et al., (2024) Journal of Agricultural and Food Chemistry, 72, 18585-18593 ). T4E functions as a C4-epimerase that directly catalyses the conversion of D-fructose to D-tagatose, a rare functional sugarwith significant industrial relevance. The T4E enzyme derived from Thermoprotei archaeon exhibit pronounced thermostability and high catalytic efficiency, making them attractive candidates for high-temperature biocatalytic processes (Chen, J. et al., (2024) Journal of Agricultural and Food Chemistry, 72, 18585-18593). Here, the thermophilic T4EP4o from Thermoprotei archaeon was immobilised onto CBSP097 matrices to assess its operational stability. Following ten repeated reaction cycles under high-temperature conditions, T4EP4o retained 68% of its initial activity, whereas the CBSP097-functionalised T4EP4o retained 90% of its initial activity (Figure 42). This result indicates a substantial improvement in enzyme stability upon immobilisation. Although the stability enhancement was somewhat less pronounced than that observed with F6PEP4o-functionalised matrices, the performance of T4EP4o on CBSP097 supports its utility for sustained, high-temperature D-tagatose production in continuous or repeated-use biocatalytic systems.

[0463] Example 17 Storage stability of BactAmyP4o-functionalised CBSP097

[0464] The storage stability of matrices functionalised with biomolecules are crucial in determining various quality parameters including the shelf-life of bioactive molecules carried by the matrices, optimum storage conditions and commercial viability. Therefore, stable functionalised matrices can reduce costs associated with production, storage, and distribution. BactAmyP4o-functionalised CBSP097 retained 100% activity after prolonged storage for 7 weeks at both 4 and 25°C (Figure 36A). At 25°C, the fluctuations in their activity between 4th and 6th week can be attributed by the variable amount of BactAmyP4o functionalised in the respective matrices (Figure 36B). However, at the end of 7 weeks both storage conditions demonstrated 100% activity relative to their initial activity (week 0). This signifies the efficacy of the proposed functionalisation technique in conferring enhanced stability to biomolecules, ensuring that they remain fully functional without degradation even at ambient temperature (25°C). This is a desirable property in various biotechnological applications.

[0465] Example 18 Comparative analysis of solubilising agents mCherryP4o was used to assess the solubilisation efficiency of several solubilising agents. Among the three different classes of solubilising agents, chaotropic salts and anionic surfactants showed substantially higher solubilisation of mCherryP4o except for L-arginine which displayed low solubilisation efficiency (Figure 37). Low and high pH buffers showed poor solubilisation efficiency except for at pH 12 which displayed almost 100% solubilising efficiency (Figure 37). When compared to Guanidine chloride (GdmCI) solubilisation (100%), Guanidine thiocyanate (GdSCN) and 2% sodium dodecyl sulfate (SDS) showed only slightly higher solubilisation of mCherryP4o. Another anionic surfactant studied was NLS, a frequently used mild solubilising agent intended to extract properly folded soluble protein from inclusion bodies (Singh, A., Upadhyay, V., Upadhyay, A. K., Singh, S. M., & Panda, A. K. (2015). Microbial Cell Factories, 14(1), 41). Upon treatment with mCherryP4o, NLS yielded 100% solubilisation, similar to GdmCI.

[0466] The influence of pH on solubilisation showed that mCherryP4o is stable and resistant to low pH, where treatment with buffers of pH 4 and 6 did not result in solubilisation while retaining their functionality. In contrast, high pH buffers showed variability in solubilisation depending on their pH strength, with solubility gradually increasing from pH 8 to pH 12. A complete solubilisation of mCherryP4o was observed when treated at pH 12, whereas treatment with pH 8 and 10 resulted only 27% and 35% solubilised mCherryP4o, respectively. This also indicated that mCherryp4o is susceptible to solubilisation at higher ionic strength pH buffers alone, without the need for chaotropic agents.

[0467] Example 19 Material functionalisation using p40-homoloqs

[0468] A preliminary experiment was performed with three selected Family AA10 p40-homologs to assess the propensity of similar protein domains within this family to form inclusion bodies (IBs) and to follow the same solubilisation-reaggregation tendency of p40. All three p40- homologs namely, KaridP4o, PsuffP4o and AlipoP4o, were expressed in E. coli as insoluble IBs (data not shown). After IBs purification, all three p40-homologs were assessed for their solubilisation- reaggregation propensity (Figure 38) using GdmCI and MilliQ waterdilution as described for p40.

[0469] GdmCI-solubilised p40-homologs (KaridP4o, PsuffP4o and AlipoP4o) at concentrations of 3.1 , 5.6 and 2 mg / mL, respectively, were used to functionalise non-woven continuous filament CBSP097 matrices. The presence of reaggregates after functionalisation was confirmed by FESEM and CLSM imaging (Figure 39 and Figure 40). Matrix functionalised with p40 served as positive functionalisation control. FESEM images of matrices functionalised with the three p40- homologs revealed a rough surface texture and presence of aggregates (similar to p40) with clumped-together adjacent fibres, indicating successful functionalisation (Figure 38). In contrast, bare matrices treated only with GdmCI retained their intact smooth surface texture.

[0470] The functionalised matrices were stained using protein dye SYPRO orange for CLSM visualisation (Figure 39). Although the bare CBSP097 matrix showed some limited SYPRO orange dye uptake, it lacked the distinct red fluorescence intensity observed in p40- and p40- homologs-functionalised matrices. The presence of bright red spots in the surface of matrix filament is due to reaggregates formed during the functionalisation process. The functionalisation of CBSP097 with AlipoP4o showed less fluorescence intensity, but this can be attributed to the lowerstarting protein concentration of AlipoP4o. All three p40 homologs were able to functionalise CBSP097 through the same solubilisation-reaggregation strategy used for p40.

[0471] Example 20 FTIR characterisation

[0472] Fourier Transform Infrared Spectroscopy (FTIR) is an indispensable technique in material science, particularly forthe functionalisation of materials. FTIR provides comprehensive insights into molecular structures, which are essential for characterising functional groups and understanding chemical modifications during functionalisation processes. Its non-destructive approach and ability to yield detailed qualitative data are crucial in confirming the molecular success of material functionalisation.

[0473] Spectral analysis of the functionalised CBSP097 material revealed the presence of amide I and amide II bands, indicating proteinaceous aggregates bound to the cellulosic fibre as a result of functionalisation (Figure 40). Since CBSP097 is a cellulosic matrix, the spectrum of bare CBSP097 showed the characteristic peaks assigned to cellulose: an absorption peak at 3348 cm'1and 2913 cm'1, corresponding to stretching vibrations of O-H and C-H bonds in cellulose. The prominent peaks at 1020 cm'1and 1051 cm'1were attributed to the C-O-C pyranose ring skeletal ring vibration. The band at 1366 cm-1 and 895 cm-1 corresponded to bending vibrations of hydroxyl group and p-glucosidic linkages between sugar moieties in cellulose, respectively. Additionally, a less intense band at 1638 cm'1, characteristic of H-O-H bending in H2O, was observed in bare CBSP097, which can be attributed to retained moisture in the matrix.

[0474] Since active inclusion bodies are known to retain secondary structure, they enable qualitative assessment using FTIR. The amide I (1600-1700 cm'1) and amide II (1500-1600 cm'1) absorption spectra are the most predominant protein characteristic spectra, originating from the C=O stretching and N-H bending vibrations of the amide group. The H2O subtracted FTIR spectrum of both mCherryP4o and BactAmyP4o -functionalised CBSP097 matrices showed the presence of two new bands which were absent in bare (non-functionalised) matrices. The absorption bands at 1654 cm'1and 1531 cm'1are attributed to the amide I and amide II groups, respectively. FTIR analysis provided definitive spectral evidence for the presence of proteinaceous aggregates within the functionalised matrices. The characteristic amide bands, indicative of protein structures, were prominently observed in the spectral data. This confirms that the material functionalisation process was successful in incorporating biomolecules, which are essential for the intended function of the matrices.

[0475] Example 21 SpinChem® RBR-mediated recycling of T4Ep40 functionalised matrices

[0476] The SpinChem® rotating bed reactor (RBR) has emerged as an efficient platform for batch and continuous enzymatic reactions using immobilised enzymes, offering advantages over stirred tank and fixed-bed reactors. The precision engineering of the SpinChem® RBR enables streamlined biocatalytic processes, wherein the RBR basket compartmentalises immobilised enzymes, separating them from the reaction mixture while remaining attached to an RPM- controlled stirrer. This setup facilitates high mass transfer, enhanced conversion efficiency, enzyme reusability, simplified downstream processing, and reduced mechanical damage to immobilised enzyme matrices (Mallin et al., (2013), ChemCatChem, 5, 3529-3532; Pithani et al., (2019), Organic Process Research and Development, 23, 1926-1931). Moreover, the RBR design enables enzyme compartmentalisation, which can be exploited for multi-enzyme cascade reactions within a single system. In this study, we employed the SpinChem® RBR to evaluate the feasibility of the present innovation for continuous biocatalytic processes at small pilot scale.

[0477] The RBR S2 basket, comprising four compartments, was loaded with T4EP4o- functionalised polypropylene (PP) fibre (Figure 43). T4EP4o-functionalised polypropylene (PP) fibres were recycled for ten cycles over approximately 24 h, with intermediate storage after the first five cycles. RBR-mediated production of D-tagatose using recycled enzyme-functionalised matrices showed retention of 31% of the initial activity at the end of ten cycles. The conversion rate decreased from 16% to 4.7% across the ten repetitive cycles, with D-tagatose yield ranging from 0.75 g / L to 0.23 g / L (Figure 44).

[0478] These findings indicate that enzyme-loaded matrices, functionalised using the present technique, are well-suited for application in the SpinChem® RBR system. Despite exposure to harsh reaction conditions, including continuous stirring at 800 RPM and elevated temperatures of 80 °C, the enzymes remained securely immobilised, with no complete desorption observed. This demonstrates the stability of the functionalised matrices under intensive mechanical and thermal stress, supporting their suitability for sustained biocatalytic processes.

[0479] Figure 45 presents the chromatogram illustrating the separation of D-fructose and D- tagatose generated via the T4EP4o-mediated reaction.

[0480] Example 22 Functionalised enzyme matrices for bioconversion of maltodextrin to D- taqatose

[0481] D-Tagatose is a rare sugar with increasing industrial relevance due to its low glycaemic index, functional health benefits, and natural sweetness, making it an attractive alternative to conventional sugars in food, pharmaceutical, and biotechnological applications. It exhibits prebiotic properties that promote gut health and is a promising ingredient fordiabetic-friendly and weight-conscious products (Roy et al., (2018), Journal of Food Science, 83, 2699-2709; Ortiz et al., (2024), Nutrients, 16, 1943). Various strategies have been explored for the biosynthesis of D-tagatose, including whole-cell biocatalysis (Dai et al., (2021), Enzyme and Microbial Technology, 145, 109747; Han et al., (2023), Journal of Agricultural and Food Chemistry, 71 , 3813-3820), and in vitro production using single enzymes (Shin et al., (2020), ACS Catalysis, 10, 12212-12222; Chen et al., (2024), Journal of Agricultural and Food Chemistry, 72, 18585- 18593), dual enzymes (Wanarska & Kur, (2012), Microbial Cell Factories, 11 , 113; Zhang et al., (2021), Biology, 10, 1343; Zheng et al., (2019), Journal of Agricultural and Food Chemistry, 67, 829-835), and multi-enzyme systems (Zhang et al., (2025), AMB Express, 15; Liu et al., (2023), Catalysts, 13, 1515; Dai et al., (2022), Biochemical Engineering Journal, 178, 108303).

[0482] Harnessing maltodextrin as a substrate in a multi-enzyme pathway offers a cost-effective and modular approach for D-tagatose production, enabling controlled enzymatic conversions. Dai et al. supra demonstrated enhanced D-tagatose production from maltodextrin using a five- enzyme pathway through modular optimisation. In earlier whole-cell approaches, co-expression of all five enzymes led to unbalanced metabolic flux, limiting overall yield and increasing reaction time. The improved strategy, involving modular expression of rate-limiting enzymes, enabled a regulated metabolic flux and resulted in a D-tagatose yield of 3.38 g / L from 10 g / L maltodextrin.

[0483] In this study, we employed a similar five-enzyme pathway for D-tagatose production from maltodextrin. All enzymes were successfully expressed in their enzymeP4o form, enabling sequential conversion of 10 g / L maltodextrin to D-tagatose. After a six-hour reaction, intact enzymeP4o fusions yielded 3.31 g / L D-tagatose, whereas conversion using functionalised enzymeP4o matrices yielded 1.13 g / L (Figure 46).

[0484] The 66% reduction in yield using functionalised matrices is attributed to enzyme loss during the functionalisation process, which likely resulted in an unbalanced enzyme ratio and gelation of maltodextrin, limiting mass transfer between enzymatic steps. Interestingly, a-glucan phosphorylase (aGP) in the functionalised system exhibited 70.5% higher glucose-1-phosphate production, yet the subsequent pathway intermediates declined sharply, ultimately limiting the overall yield. Despite this decrease, the study demonstrates the feasibility of employing functionalised enzyme matrices in multi-enzyme cascade reactions. Further optimisation is required, as this study was conducted without any prior process optimisation. The reduced yield observed with functionalised matrices is not due to limitations of the immobilisation technology itself, but rather to enzyme loss during the functionalisation process and imbalanced enzyme ratios across the cascade. These issues are likely the result of suboptimal mechanical and process parameters during the functionalisation step, such as mixing dynamics, contact time, and matrix configuration, which limited enzyme retention and led to uneven loading of enzymes. Future improvements, including fine-tuning enzyme ratios, enhancing immobilisation procedures, and co-functionalising multiple enzymes within a single matrix, will be necessary to minimise enzyme loss, maintain appropriate stoichiometry across the pathway, and ultimately enable high- yield, one-pot synthesis suitable for industrial-scale D-tagatose production.

[0485] Example 23 Computational predictions of p40’s aggregation propensity

[0486] The relative aggregating tendencies of various polypeptides can be predicted using algorithms such as AGGRESCAN (Singhvi, P., Saneja, A., Srichandan, S.and Panda, A.K. (2020) Trends in Biotechnology, 38, 474-486) and Tango (Gil-Garcia, M.and Ventura, S. (2021) Frontiers in Bioengineering and Biotechnology, 9). The Tango statistical algorithm predicts - sheet aggregation of proteins and scores above 5 % for aggregation are considered as hits (Villaverde, A., Garcia-Fruitos, E., Rinas, U., Seras-Franzoso, J., Kosoy, A., Corchero, J. and Vazquez, E. (2012) Microbial Cell Factories, 11 , 76). AGGRESCAN, on the other hand, relies on an aggregation propensity scale for each of the 20 natural amino acids obtained from in vivo experiments. Thus, AGGRESCAN prediction assumes that short and specific amino acids sequences within a protein that trigger protein aggregation. Tango has been used previously for the aggregation propensity of several protein related IBs. The co-expression of both IB forming proteins resulted in different kinetics of aggregation where the final IB mixture contains 80% of A 42-GFP which correlated to the algorithm prediction. Similarly, a study conducted by Gil- Garcia et al. detailed the use of aggregation prediction data obtained from Tango to investigate the ability of six short coiled-coil polypeptide seguences to pull down target protein (Kiisters, K., Pohl, M., Krauss, U., OIQUCU, G., Albert, S., Jaeger, K.-E., Wiechert, W.and Oldiges, M. (2021) Microbial Cell Factories, 20). p40 is a protein domain of 203 amino acid residues with a strong propensity for aggregation when expressed recombinantly in E. coli. Using two computational methods to predict protein aggregation Tango and AGGRESCAN several aggregation propensity regions for p40 can be calculated. Based on the Tango algorithm (accessed at http: / / tango.crg.es), four major aggregation-inducing regions were identified each with a length of ~10-20 amino acid (Figure. 47A). The computational prediction based on AGGRESCAN (accessed at http: / / bioinf.uab.es / aggrescan / ) identified at least six possible regions with higher aggregation propensity ranked by normalised peak area (NHSA) (Figure. 47B). Concurrently, AMYLPRED2 (accessed at http: / / biophysics.biol.uoa.gr / AMYLPRED2), a robust consensus prediction tool integrating eleven distinct algorithms, was also employed to predict aggregation propensity from the linear amino acid seguence of p40. The integration of different algorithms enhances the reliability of its predictions. The detailed output from AMYLPRED2 (Table 10) highlights the varying predictions from its constituent algorithms (9 out of 11 algorithms).

[0487] Table 10 AMYLPRED2 analysis of amyloidogenic or aggregation-prone regions in the p40.

[0488] 8-12 7-13 22-27

[0489] 7-19 11-17 7-17

[0490] 4-14 40-48 7-12 33-50 32-51

[0491] 33-45 33-42 4-10 33-51

[0492] 32-50 82-86 42-45 81-88 81-87

[0493] 90-101 91-96 39-50 81-88

[0494] 94-100 46-51 94-100 84-101 91-99 91-102

[0495] 105-116 131-138 107- 90-101

[0496] 106-113 183-188 107-113 106-114 106-114 113 105-115

[0497] 150-159 178-183 105-115

[0498] 164-171 123-127 155-156 163-172 165- 148-157

[0499] 162-172 185-190 170 162-172

[0500] 181-190 151-155 161-172 178-183 164-171

[0501] 190-196 193-198 178-196

[0502] 164-171 185-190 175-188

[0503] 190-194 While AGGRESCAN identified six broad regions, the consensus tool provides a more detailed view. For instance, WALTZ and NetCSSP appear to predict the highest number of potential aggregation-prone regions across the p40 sequence. This broader coverage by WALTZ and NetCSSP, which both utilize different scoring matrices and principles, points to distributed aggregation potential. Conversely, Tango and amyloidogenic pattern predict a considerably smaller number of aggregation-prone segments within p40 (Table 9). This divergence is expected, as each algorithm employs distinct underlying physicochemical models or empirically derived parameters to assess aggregation propensity. The consensus “hot spots’’ identified by AMYLPRED2 (7-17, 33-51 , 81-88, 90-101 , 105-115, 162-172, 178-196) represent regions with strong, consistent predictions across multiple algorithms, indicating higher confidence in their aggregation propensity. Importantly, these consensus regions comprehensively cover the aggregation-prone regions initially identified by AGGRESCAN (4-14, 32-50, 94-100, 106-113, 164-171 , 181-190). The overlap and broader range of the consensus predictions, in conjunction with AGGRESCAN's robust empirical foundation, provided sufficient confidence to proceed with AGGRESCAN-based results for guiding subsequent experimental truncated p40 variant design and characterisation.

[0504] Example 24 Construction, production and purification of different proteins fused to p40 deletion variants

[0505] The modular fusion strategy allowed feasible C-terminal incorporation of different p40 deletion variants to all the tested proteins. SDS-PAGE analysis served as the initial step to visualise the distribution of these fusion proteins, providing critical insights into their solubility and expression profiles. For each fusion combination, the lysed total cell lysate (representing all host cell proteins, including the target overexpressed protein) was compared against the supernatant (containing soluble proteins) and the insoluble fraction (indicating inclusion bodies) (Figure 48A). This enabled a direct assessment of whether the expressed proteins were predominantly soluble or insoluble. As anticipated, the majority of the p40 deletion variants, when fused to our tested proteins, led to the formation of insoluble protein fractions.

[0506] However, the consistency of these observations varied depending on the molecular weight and oligomeric state of the target protein. For instance, high-molecular-weight proteins such as monomeric a-amylase and heterotetrameric a-glucan phosphorylase (aGP), when fused with different p40 deletion partners, predominantly yielded insoluble protein (Figure 48B and C). Notably, this insolubility did not appear to compromise the overall protein yield for these larger constructs. In contrast, the fusion of these same p40 deletion variants to low-molecular-weight proteins like monomeric mCherry and homodimeric phosphoglycolate phosphatase (PGP) exhibited a more mixed distribution, with a tendency to appear in both insoluble and soluble fractions (Figure 48A and 48D). This phenomenon was observed particularly with shorter deletion variants: p40_minus 4, p40_minus 5, and p40_minus 6 when fused with mCherry. In specific the shortest deletion variant, p40_minus 6 resulted in substantial soluble fraction when fused with mCherry and PGP. Moreover, despite similar growth conditions, this variant also led to a comparatively lower overall yield for mCherry and PGP (Fig. 53B). SDS-PAGE analysis of the purified IBs from each p40 deletion construct, fused to their respective target enzymes, consistently showed bands of reduced size (Figure. 49A-D). This reduction directly corresponded to the smaller molecular weight of the p40 deletion fusion partners. This preliminary SDS-PAGE analysis provides a foundational understanding of how different p40 deletion variants influence the solubility and expression of our target proteins. While most fusions resulted in insoluble protein, suggesting a propensity for IB formation.

[0507] Example 25 Expression kinetics of mCherry fused p40 deletions

[0508] The use of mCherry fluorescent reporter allowed for direct, real-time monitoring of recombinant protein levels in E. coli and the impact of various p40 deletion variants on FIBs formation. By tracking mCherry fluorescence in vivo and utilising confocal laser scanning microscopy (CLSM), we gained insights into expression kinetics and the nature of the fusion proteins. Over a 12-hour post-induction period, cells producing different p40 deletion constructs under varying inducer (IPTG) concentrations exhibited distinct expression patterns. The control, mCherryP4o_full, showed a non-linear expression level with increasing IPTG concentrations (Figure. 50). Specifically, cells induced with 1 mM IPTG at 12 hours displayed 1.2x time decreased fluorescence compared to optimal (0.4 mM) and suboptimal (0.1 mM) IPTG concentrations. This suggests that the IBs formed at higher IPTG concentrations were larger, inherently reducing their overall fluorescence signal due to light scattering or quenching within denser aggregates. Similar observations were noted for cells producing mCherryP4o_minus 1 , minus 2. Interestingly, mCherryP4o_minus 2 showed a slightly increased fluorescence intensity (1 ,25x) across all IPTG concentrations compared to the full, minus 1 and minus 3 variants of p40 fusion. This could be attributed to their increased overall protein content and size of the IBs produced by mCherryP4o_minus 2; smaller IBs are known to contribute to enhanced fluorescence. In the case of mCherryP4o_minus 3 constructs, cells exhibited almost same fluorescence intensity at all IPTG concentrations, suggesting that the aggregation occurred at same rate irrespective of inducer concentrations.

[0509] In contrast, cells producing mCherry fused with p40_minus 4, minus 5, and minus 6 exhibited a substantially different expression pattern (Figure. 50), yielding a significant proportion of soluble mCherry with concomitantly fewer FIBs. This observation was consistent with their expression profile, as soluble mCherry produced by the p40_minus 4, minus 5, and minus 6 fusions demonstrated a 1 .5-fold increase in fluorescence over the cumulative fluorescence of the minus 1 , minus 2 and minus 3 fusions at all inducer concentration. CLSM images corroborated these findings: constructs producing functional mCherryP4o IBs (p40_full, minus 1 , minus 2, and minus 3) displayed well-defined, bright red fluorescent spots predominantly at the poles of the E. coli cells (Figure 52A) which was absent in cells containing p40_minus 4, minus 5, and minus 6. The CLSM images further evidenced this, cells expressing mCherry fused to the smaller p40 constructs (minus 4, minus 5, and minus 6) showed dispersed cytoplasmic fluorescence and dark, dense particles at the poles with weak or negligible fluorescence (Figure 52B). This was also confirmed by the SDS-PAGE analysis where cells producing mCherry fused with p40_minus 4, minus 5, and minus 6 collected at interval of post-induction (0, 2, 4 and 8 hours) showed two distinct bands one corresponding to the target mCherry alone (~26 kDa) and other the fusion protein of mCherry and corresponding p40 deletions. Although the presence of two distinct band is a contradictory observation since there is no cleavable linker between target and p40 deletion sequences, all proteins are expected to express as a single protein 38, 33.5 and 31 kDa for p40_minus 4, 5 and 6 respectively (Figure. 51 A). The presence of two distinct protein band could be attributed to cellular processes within the E. coli protein production machinery when confronted with exogenous gene expression. This can be postulated to premature translation termination or secondary structures within the mRNA encoding mCherryP4o_minus 4, minus 5, or minus 6 sequences that lead to ribosome stalling and dissociation, resulting in truncated mCherry. Regardless of the precise mechanism, these observations strongly support the finding that these shorter p40 deletion variants produce less active IBs and retain a greater proportion of mCherry in a soluble state. This clearly indicates that these smaller p40 deletion variants possess weak aggregation-inducing capacities when fused to mCherry.

[0510] Example 26 Localisation and in vivo tracking of mCherry fused p40 deletions

[0511] As previously noted, CLSM provided direct visual evidence of inclusion body (IB) formation, directly correlating with our observations from the expression kinetics. All the constructs were expressed in the same growth conditions of 0.4 mM induction for 8 hours. The presence of distinct, bright red fluorescent spots at the poles of E. coli cells was a clear indicator of active mCherry IBs formation (Figure 52). This phenomenon was consistently observed in cells expressing mCherry fused to the full-length p40, as well as the minus 1 , minus 2, and minus 3 deletion variants (Figure 52A). In contrast, cells producing mCherry fused to the shorter p40 deletion variants (minus 4, minus 5, and minus 6) showed a different localisation pattern (Figure. 52B). While these cells did exhibit distinct polar spots, these regions displayed negligible to no fluorescence. Instead, the cytoplasm of these cells was filled with uniformly dispersed red fluorescence, indicating a high yield of soluble mCherry. The big difference suggests that these shorter p40 deletion constructs were significantly less capable of inducing FIB formation with mCherry.

[0512] Example 27 Protein content estimation of different p40 deletion fusion proteins

[0513] We investigated how different p40 deletion variants affected the final yield of our target fusion protein by measuring the protein content within their respective IBs. Although all p40 deletion-based IBs were produced under identical cultivation conditions (expression at 37°C with 0.4 mM IPTG), the final protein content varied considerably. This variability appears to be directly linked to the insolubility and nature of the expressed target IBs construct. Specifically, when fused with mCherry, constructs like p40_minus 1 (88%), minus 2 (84%), and minus 4 (92%) demonstrated a 1 .3-fold increase in protein content compared to the full-length p40 fusion (73%) (Figure 53A). Interestingly, the minus 4 variant yielded the highest protein content, even though a substantial amount of active mCherry was produced in the soluble fraction (Figure. 53B and 57A) while maintaining a less active insoluble fusion protein (Figure. 53B and 57A). In contrast, the minus 3 construct fusion, while yielding functional mCherry IBs, also resulted in an equal amount of soluble protein (Figure. 53B), which consequently led to a reduced overall protein content (57%) in the IBs. Similarly, fusions with shorter deletion variants, minus 5 and minus 6, primarily produced soluble mCherry. This solubility was reflected in their lower final IB protein content, with inactive mCherry IBs from these constructs containing 58% and 61 % of the target protein, respectively.

[0514] Observations reveal that the influence of p40 deletion variants on target protein yield is highly specific to the target protein itself. The trends observed with mCherry fusion proteins are not consistent with other targets, such as the enzyme targets a-amylase and aGP. These enzymes demonstrated a positive effect on final yield or protein content when fused with various p40 deletion variants. For instance, functionally active a-amylase IBs produced from all p40 fusion variants consistently exhibited comparatively higher protein content. The minus 3 fusion yielded a maximum of 102% protein content, while the minus 1 fusion showed a minimum of 80% when compared to the full-length p40 fusion, which comprised 96% target IBs. Other a-amylase fusions resulted in protein content between 83% and 94% (Figure. 53B). In the case of aGP, the two tested variants, minus 3 and minus 4, contained nearly identical protein amounts, ranging from 61 % to 69%. However, when these same deletion constructs were fused to PGP, minus 3 resulted in a similar protein amount compared to full-length p40 (86%), whereas minus 4 led to a 1.4-fold decreased yield (62%). This reduction with the minus 4 variant was attributed to a considerable amount of PGP produced as soluble fraction. Therefore, the impact of p40 deletion fusions on target protein content is target protein-specific. This suggests that the sequence features of both the p40 deletion variant and the target protein play a crucial role in determining the final yield. These fusion partners could potentially be utilised to achieve tunable yields of target protein IBs.

[0515] Example 27 Influence of p40 deletions in the size of IBs

[0516] The biophysical characteristics of IBs, including their size, shape, and surface texture, can be uniquely influenced when IBs are produced by fusing different pull-down tags or fusion partners. Field Emission Scanning Electron Microscopy (FESEM) is invaluable for detailed morphological analysis of these IBs, providing a thorough understanding of how various fusion tags impact these critical physical attributes such as size, shape and surface texture. This section details the FESEM-based analysis of IBs generated with different p40 deletion variants. Despite identical growth conditions, the fusion of distinct p40 deletion variants demonstrably impacted the size and morphology of the resulting IBs. All studied target protein IBs, mCherry, a-amylase, aGP, and PGP when fused to p40 deletion variants, exhibited comparative size differences relative to full-length p40, generally showing a decrease in size with shorter deletion variants. This suggests that specific sequence determinants or "hot spots" within the p40 deletion not only trigger IB formation but also influence the physical properties of the final IBs. This finding highlights the potential of these p40 fusion partners fortunable IB production.

[0517] For example, all mCherry IBs obtained from different p40 deletion variants maintained a consistent spherical morphology (Figure. 54), but their sizes varied. The maximum IB bead diameter was 0.608 microns, produced by the p40_minus 2 fusion, while the minimum was 0.538 microns, observed with the p40_minus 6 fusion. Other p40 fusion constructs yielded mCherry IBs with diameters falling within the range of 0.551 to 0.604 microns (Table 11).

[0518] Table 11 Size distribution of mCherryP4o IBs produced by fusing different p40 deletion variants

[0519] FESEM mCherryP« deletions Diameter (pm) SD full 0.604 ± 0.005 0.232 ± 0.029 minus 1 0.582 ± 0.008 0.156 ± 0.025 minus 2 0.608 ± 0.005 0.178 ± 0.017 minus 3 0.576± 0.006 0.172 ± 0.022 minus 4 0.593 ± 0.009 0.216 ± 0.039 minus 5 0.551 ± 0.010 0.213 ± 0.039 minus 6 0.538 ± 0.006 0.180 ± 0.024 In contrast to the consistent spherical morphology of mCherry IBs, BactAmy IBs resulting from various p40 deletion fusions displayed mixed morphological and size distributions. Fusions with full-length p40, minus 1 , minus 2, and minus 5 variants led to the formation of elongated, cylindrical IB structures (Figure. 55). Each of these beads had a rough surface, likely formed by the agglomeration of insoluble protein aggregates. This resulted in a highly non-homogenous size distribution, with IBs ranging from 0.694 to 0.752 microns (Table 12). Interestingly, fusions with the minus 3, minus 4, and minus 6 p40 deletion variants produced spherical IBs of smaller size, ranging from 0.606 to 0.638 micrometers (Table 12). These observed differences in the final IB size and morphology can be attributed to the conformational features of the respective p40 deletion variants and BactAmy itself. These combined features appearto influence the nucleation of IB formation, ultimately leading to the varied morphological structures observed.

[0520] Table 12 Size distribution of BactAmyp40 IBs produced by fusing different p40 deletion variants

[0521] FESEM

[0522] BactAmyP4o deletions Diameter

[0523] SD full 0.752±0.18 0.219 ±0.060 minus 1 0.725 ±0.14 0.181 ±0.035 minus 2 0.74 ±0.13 0.174 ±0.030 minus 3 0.606 ±0.08 0.105 ±0.020 minus 4 0.608±0.08 0.110 ±0.020 minus 5 0.694 ±0.1 0.154 ±0.045 minus 6 0.638 ±0.08 0.104 ±0.020

[0524] To further investigate how p40 deletion variants influence IB size, the two best-performing variants, p40_minus 3 and p40_minus 4 were selected. These variants were chosen due to their shorter sequence lengths and their ability to produce functional IBs (FIBs). They were then fused to two additional targets with higher oligomeric states: aGP (heterotetramer) and PGP (homodimer). Functional IBs from these new fusions consistently exhibited smaller sizes compared to those produced with full-length p40. For aGP, the full-length p40 fusion produced IBs with a diameter of 0.883 microns. In contrast, the minus 3 and minus 4 fusions resulted in smaller IBs of 0.761 and 0.643 microns, respectively (Table 12). Notably, all aGPP4o IBs from these fusions displayed a smooth surface (Fig. 56A-C). Similarly, the fusion of full-length p40 to PGP produced IBs with a size of 0.62 microns. A further reduction in size was observed with the shorter p40 deletions: minus 3 and minus 4 fusions yielded PGP IBs of 0.553 and 0.40 microns, respectively (Table 13). FESEM images indicated that the nature of PGPP4o IBs was significantly influenced by the fusion partner. While the full-length p40 fusion produced well-defined spherical IBs (Fig. 56D), PGPP4o IBs from the minus 3 and minus 4 fusions appeared as soft aggregates with indefinite shapes (Figure. 56E and 56F). This was particularly true for the p40_minus 4 fusions, which also yielded a substantial amount of soluble PGP, likely contributing to the formation of these soft, less defined aggregates.

[0525] Table 13 Size distribution of mCherryP4o IBs produced by fusing different p40 deletion variants.

[0526] FESEM p40 deletions Diameter (pm) SD aGPP4o full 0.883±0.141 0.175±0.035 aGPP4o minus 3 0.761 ±0.096 0.131±0.026 aGPP4o minus 4 0.643±0.083 0.111±0.022

[0527] PGPP4o full 0.62±0.126 0.160±0.031

[0528] PGPP4o minus 3 0.553±0.010 0.131±0.037

[0529] PGPP4o minus 4 0.401±0.090 0.110±0.031

[0530] In conclusion, FESEM analysis definitively shows that the p40 deletion variants exert a profound and specific influence on the biophysical characteristics of IBs, directly impacting their size, shape, and surface texture. This demonstrates a powerful tunability in IB morphology, dictated not only by the fusion partner but critically by the specific p40 deletion. The observed variations in IB structure, from defined spheres to soft aggregates, underscore the potential to engineer IBs with tailored properties for diverse functional applications.

[0531] Example 28 Retained functionality of different p40 deletion variants

[0532] Having explored the production and biophysical characteristics of various target protein IBs generated by fusion with distinct p40 deletion variants, it's crucial to examine how these fusions influence the functionality of the resulting IBs. All six deletion variants were fused to two targets, mCherry and BactAmy and the respective activity was compared with the IBs produced by full length p40 fusion. For mCherryP4o IBs, retained fluorescence served as a direct measure of bioactivity. The results clearly demonstrate that different p40 deletion constructs significantly influenced the final fluorescence of mCherryP4o IBs. The maximum fluorescence was observed for mCherry fused to the minus 2 variant, which was 1 .2 times higher than that of the full-length p40 fusion. This increase is primarily attributed to the enhanced protein content of the mCherryP4o minus 2 IBs. Interestingly, despite the minus 1 fusion yielding higher protein content among other fusions, this did not correlate with its retained fluorescence, which showed a 1.4-fold decrease compared to the minus 2 variant (Figure. 57A). This apparent contradiction likely arises from conformational changes induced by the fusion of specific p40 deletion variants, influencing chromophore formation within the mCherryP4o IBs. Furthermore, retained fluorescence began to decrease with fusions from minus 4 to minus 6. This decline is largely attributed to the substantial soluble expression of mCherry with these variants, resulting in a less active fraction being incorporated into the IBs.

[0533] In contrast to mCherry, BactAmy IBs produced by fusion with all six p40 deletion variants were fully expressed as IBs. However, their catalytic activity was notably affected by certain p40 deletion fusions (Figure.57B). Except forthe shorter variants, minus 5 and minus 6, all other p40 fusions resulted in comparable amylase activity. The minus 5 and minus 6 fusions, conversely, exhibited only 29-42% of the initial activity by fusion of full length p40. Given that the protein content of these constructs was not significantly impacted, this decline in activity likely resulted from the formation of less active IBs after the fusion. Interestingly, the minus 3 and minus 4 variants showed a pronounced increase in activity, approximately 1 .6-fold higher than the full- length p40 fusion. This enhanced activity correlates with their protein content. Furthermore, FESEM images revealed that BactAmyP4o IBs from the minus 3 and minus 4 fusions were smaller in size. This reduced size could significantly enhance the mass transfer of substrate to the catalytic sites within the IBs, thereby contributing to their higher activity.

[0534] These observations align with similar findings in the literature regarding the influence of fusion tags on the catalytic activity of inclusion bodies. For instance, Jager et al. (Jager, V.D., Kloss, R., Griinberger, A., Seide, S., Hahn, D., Karmainski, T., Piqueray, M., Embruch, J., Longerich, S., Mackfeld, U., Jaeger, K.-E., Wiechert, W., Pohl, M.and Krauss, U. (2019) Microbial Cell Factories, 18, 33) demonstrated that the fusion of two different coiled-coil domains, 3HAMP and TDoT, as pull-down tags significantly impacted the catalytic efficiency of resulting catalytically active IBs (CatIBs). Despite identical production parameters, CatIBs from the 3HAMP fusion consistently outperformed TDoT CatIBs, a difference that correlated with the protein content ofthe respective CatIBs. In another example, three synthetic fusion tags (18AWT, L6KD, GFIL8), all comprising smaller residues (0.9-2.3 kDa), were evaluated for their efficiency in producing CatIBs with alcohol dehydrogenase from Ralstonia sp. (RADH), an industrially important enzyme. Among the three tags, N-terminal fusion of GFIL8 exhibited a 3-fold increase in RADH catalytic activity compared to the other tested tags, strongly indicating that both the choice and position of fusion tags are critical determinants of CatIBs1intrinsic catalytic activity. These consistent observations, particularly with the enzyme-p40 IBs produced by fusion of p40 deletion variants, imply that the retained catalytic activity of the resulting IBs is highly dependent on the conformational features of the fusion partners. These features, in turn, dictate the fate of the misfolded protein aggregates, highlighting that most of the p40 deletion variants can serve as successful candidates for producing tunable CatIBs. Therefore, careful selection of p40 deletion variants provides an effective means to fine-tune the biophysical properties and catalytic performance of resulting IBs.

[0535] Example 29 Ability of different p40 deletions-based enzyme IBs to functionalise matrix and their retained activity

[0536] In the previous examples, we exploited the intrinsic ability of p40-based IBs to reversibly aggregate upon chaotropic solubilisation and stepwise dilution. This property was exploited to demonstrate an innovative material biofunctionalisation strategy. To further extend this, the same approach was tested using IBs produced by fusing different p40 deletion variants. Three enzymes with varying folding complexities were chosen as model proteins: BactAmy (monomer), PGP (homodimer), and aGP (heterotetramer). Equivalent amounts of each enzyme IB from all deletion variants were used for material biofunctionalisation, and their relative activity was compared with that of their respective IBs produced by full length p40 fusion. CBSP097, a nonwoven cellulose and PET multilayer matrix functionalised with BactAmyP4o IBs, showed a similar activity trend to that observed for the intact IBs. Specifically, matrices functionalised using the p40_minus 3 and minus 4 fusions exhibited higher activity (110 and 114% respectively) than those functionalised with full-length p40. Conversely, fusions from minus 5 and minus 6 resulted in lower activity (13 and 23% respectively), showing the same observations for intact IBs (Figure 58A). Despite showing the same trend in retained activity, a slight difference in the relative activity of respective BactAmyP4o after functionalisation was noted. This can be attributed to the loss of enzyme during the functionalisation process, leading to varying concentrations of BactAmyP4o retained in the matrices and subsequently decreased activity. Similar observations were made with matrices functionalised with aGPP4o. In their intact form, all p40 deletion fusions exhibited comparatively similar activity (100%) to full-length p40. However, after functionalisation, aGP_minus 3 and minus 4 variants retained 95% and 89% of their initial activity, respectively, relative to intact IBs (Figure. 58B). The slight decrease in activity could be due to enzyme loss during functionalisation. This indicates that despite being shorter p40 variants, minus 3 and minus 4 demonstrated enhanced efficiency in forming CatIBs while retaining the crucial ability for reversible aggregation. The fusion of these deletion variants with another target, PGP, also yielded similar observations. CBSP097 functionalised with PGPP4o_full and minus 3 showed activity comparable to their respective intact IBs (Figure. 58C). Interestingly, the matrix functionalised with PGPP4o_minus 4 exhibited a 1.6-fold increased activity compared to its intact IB counterpart. This anomaly might be attributed to batch variation in the enzyme preparation for PGPP4o_minus 4.

[0537] This investigation demonstrates that p40 deletion variants that possess distinct sequences determinants not only capable of producing CatIBs but also retained biofunctionalisation capabilities. The observed trends in retained enzymatic activity on solid matrices largely reflected the intrinsic activity profiles of the intact IBs, confirming that the properties conferred by the p40 fusion are maintained upon functionalisation. Notably, p40_minus 3 and minus 4 variants consistently proved effective in forming CatIBs that retained their reversible aggregation properties, making them promising candidates for robust material functionalisation.

[0538] Examples 24 to 29 described herein demonstrate a rational approach to identifying shorter aggregation-prone tags within the larger p40 aggregation-inducing protein domain. Utilising computational predictions, six p40 deletion variants, each containing at least one aggregation “hot spot’’, were designed and evaluated for their ability to produce FIBs. These variants were fused to four diverse protein targets representing varying degrees of complexity: mCherry (a monomeric fluorescent protein), a-amylase (a monomeric enzyme), PGP (a homodimeric enzyme), and aGP (a heterotetrameric enzyme). Preliminary result showed the fusion of deletion variants with mCherry and a-amylase targets successfully produced functional IBs (FIBs). However, there is a strong influence of certain deletion variants in theiryield, physical properties, and functionality. Based on their shorter sequence length and retained aggregationinducing capabilities, p40_minus 3 and p40_minus 4 deletion variants were identified as the most promising candidates. These were subsequently fused to the more complex enzyme targets, aGP and PGP, to further investigate their impact on IB yield, size, and catalytic efficiency. This comprehensive analysis revealed that these deletion fusion partners consistently produced FIBs with tailored characteristics. In summary, while the full-length p40 domain has already proven its utility in producing stable IBs for various biotechnological applications, the identification of shorter, functional aggregation-inducing partners within p40 offers significant advantages. This not only contributes to deciphering the molecular basis of protein aggregation but also facilitates the development of refined fusion tags capable of producing FIBs with tunable properties. Furthermore, these p40 deletion variants retained the ability for reversible aggregation, similarto full-length p40 domain. This strongly implies that the underlying mechanism of reversible aggregation and subsequent functionalisation is driven by the specific sequence determinants present within these refined p40 deletions and the fused target proteins themselves. Therefore, rational approach to identify aggregation-inducing tags within the larger aggregation-inducing protein domain offers a promising toolbox approach to develop diverse fusion partner for producing FIBs with tunable properties for various biotechnological and biomedical applications.

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[0547]

Claims

CLAIMS:1 . A functionalised matrix comprising:(i) a protein nanoparticle (PNP) having an aggregating part capable of forming or aggregating into a protein particle and a functional part providing functional activity to the matrix; and(ii) a porous matrix; wherein the PNP is absorbed, combined, bound to, or contained within the porous matrix.

2. The matrix according to claim 1 , wherein the PNP is absorbed, combined, bound to or contained within the porous matrix following solubilisation of the PNP and reaggregation in the presence of the matrix.

3. The matrix according to claim 1 or 2, wherein the PNP is an insoluble protein aggregate.

4. The matrix according to any one of claims 1 to 3, wherein the PNP is solubilised with a solubilising agent that permits solubilisation of the PNP without causing denaturation of the PNP.

5. The matrix according to any one of claims 1 to 4, wherein the solubilising agent is guanidinium chloride (GdmCI).

6. The matrix according to any one of claims 1 to 5, wherein reaggregation occurs once combined with the matrix.

7. The matrix according to any one of claims 1 to 6, wherein the aggregating part is a synthetic peptide or a naturally occurring peptide.

8. The matrix according to any one of claims 1 to 7, wherein the aggregating part of the PNP is selected from an Auxiliary Activity Family 10 (AA10) enzyme.

9. The matrix according to any one of claims 1 to 8, wherein the aggregating part is a protein comprising or consisting of a sequence or functional part thereof set forth in Table 1 .

10. The matrix according to any one of claims 1 to 9, wherein the aggregating part is a protein comprising a sequence or functional part thereof selected from a sequence set forth in SEQ ID NOs: 1-4 or 27-124.

11. The matrix according to any one of claims 1 to 10, wherein the aggregating part of thePNP is p40 or an analogue thereof.

12. The matrix according to any one of claims 1 to 11 , wherein the aggregating part comprises or consists of a p40 sequence set forth in: VFPATRTYACYVDGKVHGNGGDLNMINPACLDALAISGNYQFWNWFGNLISNAGGRHREIIPD GKLCGPTASFDGMNQARTDWWTTRLQPGATITVRVNAWAPHPGTWYLYVTRDGWDPTQPL KWSDLEPTPFSQVTNPPINSSGPDGAEYSWQVQLPNKQGRHIIYMIWQRSDSPEAFYNCSDV YFGSGPIAYEFGDPREGG (SEQ ID NO:1) or a sequence at least 60% identical thereto.

13. The matrix according to any one of claims 1 to 12, wherein the p40 protein comprises or consists of a sequence at least 65% at least 67%, at least 70%, at least 72%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 98% or at least 99% identical to the sequence of SEQ ID NO:1.

14. The matrix according to any one of claims 1 to 13, wherein the aggregating part comprises a p40 sequence set forth in SEQ ID NO:1 .

15. The matrix according to any one of claims 1 to 13, wherein the aggregating part comprises a p40 sequence set forth in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

16. The matrix according to any one of claims 1 to 15, wherein the p40 sequence is a p40 fragment comprising a sequence of SEQ ID NOs:21 to 26.

17. The matrix according to claim 16, wherein the p40 fragment is SEQ ID NO:24 or 25.

18. The matrix according to any one of claims 1 to 17, wherein the functional part of the PNP is selected from mCherry, enhanced green fluorescence protein (eGFP), alpha-amylase (BactAmy), alpha glucan phosphorylase (aGP) or phosphoglycolate phosphatase (PGP), phosphoglucomutase (PGM), glucose-6-phosphate isomerase (PGI), beta-galactosidase ( - Gal), frutalin (FTL), microvirin (MVN), metallothionein (metallo), anti-His6 nanobody (Anti-His), ZXR-2 , BactXylB, F6PE, fructose 6-phosphate 4-epimerase (F6PE) and Tagatose 4-epimerase (T4E).

19. The matrix according to any one of claims 1 to 17, wherein the functional part of the PNP comprises an enzyme, peptide, a lectin, an antigen-binding molecule, a metallothionein, a fluorescent label or other biomolecule.

20. The matrix according to any one of claims 1 to 17, wherein the functional part of the PNP comprises a red or green fluorescence, a hydrolase, a phosphorylase, a glycosidase, or an isomerase.21 . The matrix according to any one of claims 1 to 20, wherein the functional part is capable of binding to or being bound by a target compound.

22. The matrix according to any one of claims 1 to 20, wherein the aggregating part and the functional part are fused together.

23. The matrix according to any one of claims 1 to 20, wherein the aggregating part and the functional part are joined by a linker.

24. The matrix according to any one of claims 1 to 23, wherein the PNP comprises one or more different functional parts capable of binding one or more different target components.

25. The matrix according to any one of claims 1 to 24, wherein the functionalised matrix comprises two or more different p40 proteins.

26. The matrix according to any one of claims 1 to 25, wherein the PNP comprises:(i) the sequence of SEQ ID NO:5;(ii) the sequence of SEQ ID NO:6;(Hi) the sequence of SEQ ID NOT;(iv) the sequence of SEQ ID NO:8;(v) the sequence of SEQ ID NO:9;(vi) the sequence of SEQ ID NO:10;(vii) the sequence of SEQ ID NO:11 ;(viii) the sequence of SEQ ID NO:12;(ix) the sequence of SEQ ID NO:13;(x) the sequence of SEQ ID NO:14;(xi) the sequence of SEQ ID NO:15;(xii) the sequence of SEQ ID NO:16;(xiii) the sequence of SEQ ID NO:17;(xiv) the sequence of SEQ ID NO:18;(xv) the sequence of SEQ ID NO: 19; or(xvi) the sequence of SEQ ID NO:2027. The matrix according to any one of claims 1 to 26, wherein the functionalised matrix is recyclable or reusable.

28. A method of forming a functionalised matrix, the method comprising:(i) producing a protein nanoparticle (PNP) having an aggregating part capable of forming or aggregating into a protein particle and a functional part providing functional activity to the matrix; and’(ii) combining the PNP with a matrix wherein the PNP is in a solubilised form and reaggregation occurs in the presence of the matrix such that the PNP is absorbed, combined, bound to, or contained within the matrix.

29. A method of forming a functionalised matrix, the method comprising:(i) harvesting insoluble protein aggregates, the aggregates comprising a protein nanoparticle (PNP), the PNP having an aggregating part capable of forming or aggregating into a protein particle and a functional part providing functional activity to the matrix;(ii) solubilising the insoluble protein aggregates to yield the PNP;(iii) combining the solubilised PNP with a matrix; and(iv) allowing the solubilised PNP to reaggregate in the presence of the matrix for a time sufficient to allow the PNP to be absorbed, combined, bound to or contained within the matrix.

30. The method according to claim 28 or 29, wherein the PNP is capable of being solubilised without denaturing.

31. The method according to any one of claims 28 to 30, wherein the method comprises solubilising the insoluble protein aggregates with guanidinium chloride (GdmCI).

32. The method according to any one of claims 28 to 31 , wherein the matrix is a porous matrix.

33. The method according to any one of claims 28 to 32, further comprising providing a nucleic acid molecule encoding the PNP to a cell and allowing the cell to express the nucleic acid molecule to form an insoluble protein aggregate and recovering the insoluble protein aggregate.

34. The method according to any one of claims 28 to 33, wherein the PNP is cloned into a vector which is expressible in a cell.

35. The method according to any one of claims 28 to 34, wherein the functional part of the PNP is derived from a bacterium.

36. The method according to claim 35, wherein the bacterium is selected from the group consisting of Anaplasma marginale, Aequorea Victoria, Bacillus licheniformis, Bacillus subtilis, Dictyoglomus turgidum, Thermus Thermophilus, Archaeoglobus profundus, Artocarpus incisa, Microcystis aeruginosa, Pisum sativum, Sulfolobus acidocaldarius and Androctonus mauritanicus.

37. The method according to any one of claims 28 to 35, wherein the functional part of the PNP is selected from mCherry, enhanced green fluorescence protein (eGFP), alpha-amylase (BactAmy), alpha glucan phosphorylase (aGP) or phosphoglycolate phosphatase (PGP), phosphoglucomutase (PGM), glucose-6-phosphate isomerase (PGI), beta-galactosidase ( - Gal), frutalin (FTL), microvirin (MVN), metallothionein (metallo), anti-His6 nanobody (Anti-His), ZXR-2, BactXylB, F6PE, fructose 6-phosphate 4-epimerase (F6PE) and Tagatose 4-epimerase (T4E).

38. The method according to any one of claims 28 to 35, wherein the functional part of the PNP may comprise protein A, protein G, protein L, an antigen binding molecule (e.g. antibody or aptamer), a single chain antibody, avidin, streptavidin, an enzyme, an inhibitor, an antigenic determinant, an epitope, a binding site, a lectin, a cellulose binding protein, a polyhistidine, an oligohistidine, a receptor, a hormone, a signalling molecule, a polypeptide with specific or group specific binding capabilities, or a combination thereof.

39. The method according to any one of claims 28 to 35, wherein functional part of the PNP may comprise an enzyme, peptide or a fluorescent label.

40. The matrix or method according to any one of claims 1 to 39, wherein the matrix comprises a material which is capable of being functionalised.41 . The matrix or method according to any one of claims 1 to 40, wherein the matrix material is a natural or synthetic zeolite, silica, glass wool, metal-based fibre, a pad, or porous bead.

42. The matrix or method according to any one of claims 1 to 40, wherein the matrix material is selected from a polypropylene, a cellulose, a cupro / polyethylene terephthalate (PET), PET microfibre, PET, a polyethylene, a methacrylate, a dextran or polystyrene.

43. The matrix or method according to any one of claims 1 to 42, wherein reaggregation of the solubilised PNP occurs in the presence of the matrix.

44. The matrix or method according to any one of claims 1 to 43, wherein the solubilisation and reaggregation of the PNP and matrix comprises agitation.

45. A method for separating at least one target component or compound from a sample or mixture comprising the target component or compound, the method comprising:(i) combining the functionalised matrix comprising the PNP according to any one of claims 1 to 27, or prepared according to the method of any one of claims 28 to 44, with a sample or mixture containing the target component or compound; and(ii) allowing a target component in the sample or mixture to bind to the functional part of the PNP.

46. A method for enriching or separating at least one target component or compound from a sample or mixture comprising the target component or compound, the method comprising combining the functionalised matrix comprising the PNP according to any one of claims 1 to 27, with a sample or mixture containing the target component or compound whereby enriching the at least one target component or compound from the sample or mixture.

47. The method according to claim 45 or 46, wherein the mixture comprises a suspension, dispersion, solution or combination thereof of any biological extracts or derivatives thereof.

48. The method according to any one of claims 45 to 47, wherein the target component is selected from a protein, a peptide, a polypeptide, an immunoglobulin, biotin, an inhibitor, a cofactor, a substrate, an enzyme, a receptor, a monosaccharide, an oligosaccharide, a polysaccharide, a glycoprotein, a lipid, a nucleic acid, a cell or fragment thereof, a cell extract, an organelle, a virus, a biological extract, a hormone, a serum protein, a milk protein, a milk-derived product, blood, serum, plasma, a fermentation product a macromolecule or any other molecule or any combination or fraction thereof.

49. The matrix according to any one of claims 1 to 27, wherein the functionalisation efficiency ofthe functionalised matrix is greaterthan or equal to 85%, greaterthan or equal to 87%, greater than or equal to 90%, greater than or equal to 92%, greater than or equal to 95%, greater than or equal to 97%, greater than or equal to 99% or 100%.

50. The matrix according to any one of claims 1 to 27, wherein the enzyme activity of the functionalised matrix is greater than 100%, greater than 110%, greaterthan 120%, greater than130%, greater than 140%, greater than 150%, greater than 160% or greater than 170% compared to its activity prior to functionalisation.51 . The matrix according to any one of claims 1 to 27, for use or when used in a method of medical treatment.

52. A kit for affinity separation comprising the functionalised matrix according to any one of claims 1 to 27 and instructions for carrying out affinity separation.

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