Polymer composite for enzyme immobilization and methods thereof

Abstract

The present invention relates to a polymer composite, comprising a matrix formed from polyacrylate, polyethylene glycol (PEG) and ionisable surfactant; wherein the polyacrylate at about 9 wt% to about 20 wt% relative to the polymer composite; wherein the polyethylene glycol is electrostatically bonded to the polyacrylate, and at about 70 wt% to about 90 wt% relative to the polymer composite; and wherein the ionisable surfactant is homogenously dispersed within the matrix, and at about 1 wt% to about 10 wt% relative to the polymer composite.

Description

[0001] Polymer Composite for Enzyme Immobilization and Methods Thereof

[0002] Technical Field

[0003] The present invention relates, in general terms, to a polymer composite for enzyme immobilization, the method of fabrication and uses thereof.

[0004] Background

[0005] Without immobilization, standalone enzymes face the technical challenge of losing activity quickly due to environment, e.g., heat or pH values. Furthermore, the standalone enzyme is not applicable for reuse (e.g., continuous cycled process) or recovery, may quickly lose activity, and need to be removed through purification after reaction, causing production cost increase.

[0006] Many studies detail different techniques for immobilizing biomolecules in general and enzymes in specific. Enzyme immobilization's primary objectives are still to significantly limit an enzyme's freedom of movement, lower an enzyme's cost contribution throughout the process, enable heterogeneous catalysis of enzymatic reactions and operation under continuous processes, and prevent the need to extract an enzyme from the final product.

[0007] Numerous methods have been used to immobilize enzymes and other macromolecules. One of these processes is the physical adsorption of enzymes to solid, insoluble carrier matrices such polymers, silica, celite, and glass beads. Method of adsorption on ion-exchange resins or to a solid carrier has also been used, and further cross-linking with bi-functional molecules, such as glutaraldehyde is sometimes applied.

[0008] Other than adsorption, covalent binding to a solid material, (e.g., activated silica) has also been used.

[0009] Entrapment of enzymes in polymers, (e.g., acrylic-based polymers),encapsulation of enzymes in gels or confinement of enzyme in a membrane reactor have also been applied.

[0010] Another common method involves the cross-linked enzyme crystals or aggregates.

[0011] In all these methods, problems associated with enzyme immobilisation includes reduced enzyme activity due to conformational changes, steric hindrance, or reduced accessibility of the active site, mass transfer limitations where the substrate and product have difficulty penetrating the immobilisation matrix, leakage of the enzyme, reduced operational stability as the immobilisation process can make the enzyme more susceptible to denaturation, inactivation, or degradation, and increased cost.

[0012] The immobilization materials further need to possess good mechanical properties, high loading and tailorability for specific activity.

[0013] It would be desirable to overcome or ameliorate at least one of the abovedescribed problems.

[0014] Summary

[0015] The present disclosure provides a polymer composite, comprising a matrix formed from polyacrylate, polyethylene glycol (PEG) and ionisable surfactant; wherein the polyacrylate at about 9 wt% to about 20 wt% relative to the polymer composite;

[0016] wherein the polyethylene glycol is electrostatically bonded to the polyacrylate, and at about 70 wt% to about 90 wt% relative to the polymer composite; and wherein the ionisable surfactant is homogenously dispersed within the matrix, and at about 1 wt% to about 10 wt% relative to the polymer composite.

[0017] In some embodiments, the polyacrylate is selected from poly(methacrylate), polymethyl methacrylate, polyethyl methacrylate, poly(2-hydroxylethyl methacrylate).In some embodiments, the polyacrylate is characterised by a molecular weight of about 10,000 g / mol to about 1,000,000 g / mol.

[0018] In some embodiments, PEG is characterised by a molecular weight of about 100 g / mol to about 1000 g / mol.

[0019] In some embodiments, a molecular weight ratio of polyacrylate to PEG is about 1000 to about 3000.

[0020] In some embodiments, a weight ratio of polyacrylate to PEG is about 1:10 to about 4:10.

[0021] In some embodiments, the ionisable surfactant is ionised to a positive or negative charge.

[0022] In some embodiments, the ionisable surfactant is selected from tetraalkylammonium salt or long chain fatty acid or salt thereof.

[0023] In some embodiments, the ionisable surfactant is selected from stearic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid and tetrabutylammonium salt, tetrapropylammonium salt, alkylbenzyldimethylammonium salt, distearyldimethylammonium salt, cetrimonium salt and behentrimonium salt.

[0024] In some embodiments, a weight ratio of polyacrylate to ionisable surfactant is about 1:1 to about 5:1.

[0025] In some embodiments, a weight ratio of polyacrylate to PEG to ionisable surfactant is 15:80:5.

[0026] In some embodiments, the polymer composite further comprising carbon nanoparticles.In some embodiments, the carbon nanoparticle is about 0.01 wt% to about 1 wt% relative to the polymer composite.

[0027] In some embodiments, the polymer composite is characterised by a particle size of about 100 pm to about 600 pm.

[0028] In some embodiments, the polymer composite is characterized by a first endothermic peak temperature of about 65 °C to about 85 °C.

[0029] In some embodiments, the polymer composite is characterized by a second endothermic peak temperature of about 90 °C to about 115 °C.

[0030] In some embodiments, the polymer composite is characterised by a degree of crystallinity of about 1% to about 20%.

[0031] In some embodiments, the polymer composite is characterized by a pore size distribution of about 8 nm to about 250 nm.

[0032] In some embodiments, the polymer composite is characterized by a Vpore / V-total of more than about 80%.

[0033] The present disclosure also provides an immobilised enzyme, wherein the enzyme is immobilised on a polymer composite as disclosed herein.

[0034] In some embodiments, the enzyme is adsorbed on the polymer composite.

[0035] In some embodiments, the enzyme is selected from allulose isomerase, lipase, esterase, cutinase, PETase, or a combination thereof.

[0036] In some embodiments, the immobilised enzyme is characterised by an enzyme activity of at least 6 times relative to a free enzyme.

[0037] In some embodiments, when the polymer composite comprises positively ionisable surfactant and carbon nanoparticles, the immobilised enzyme ischaracterised by an enzyme activity of at least 5 times relative to an enzyme immobilised on a polymer composite without carbon nanoparticles.

[0038] The present disclosure also provides a method of fabricating a polymer composite, comprising:

[0039] a) dissolving polyacrylate, polyethylene glycol and ionisable surfactant in a solvent to form a solution; and

[0040] b) mixing the solution with a non-solvent in order to precipitate the polyacrylate, polyethylene glycol and ionisable surfactant to form the polymer composite.

[0041] In some embodiments, step a) further comprises adding carbon nanoparticles to the solution.

[0042] Brief description of the drawings

[0043] Embodiments of the present invention will now be described, by way of nonlimiting example, with reference to the drawings in which:

[0044] Figure 1 illustrates hydrogen bonding between PMMA and PEG. Green dash lines are the indication of hydrogen bonding.

[0045] Figure 2 shows representative SEM images for A: PMMA resin with stearic acid; B: PMMA resin with stearic acid & Carbon NPs; C: PMMA resin with TBABiS; D: PMMA resin with TBABiS & Carbon NPs.

[0046] Figure 3 shows loading of enzyme DM1-514 onto resins Ex. 1 and Ex. 2. Columns 1 and 3 are the bounded enzyme, while columns 2 & 4 are for the nonbounded enzyme.

[0047] Figure 4 shows loading of enzyme DM1-514 onto resins Ex. 3 and Ex. 4. columns 1 and 3 are the non-bounded enzyme and columns 2 and 4 are for the bounded enzyme.

[0048] Figure 5 shows Ih activity for immobilized enzyme DM1-514, Ex. 3-i: enzyme immobilized onto Ex. 3, Ex. 4-i: enzyme immobilized onto Ex. 4, Ex. 5-i: enzyme immobilized onto Ex. 5, Ex. 6-i: enzyme immobilized onto Ex. 6.Detailed description

[0049] The present disclosure is based on the understanding that in immobilization of enzyme through non-covalent adsorption, identification of an appropriate support is very important. Characteristics of the support material, such as high affinity for enzyme / protein, rigidity, and mechanical stability should be carefully taken into consideration.

[0050] In this regard, polyacrylate is a class of polymer with acrylate (e.g., methyl methacrylate) backbones and is hydrophobic. This class of polymer and its composite have good resistance to mechanical stress, such as flow and friction, making them a good group of candidates for enzyme immobilization purpose.

[0051] In some embodiments, the present disclosure relates to an acrylic resin composite consisting of backbone of polyethylene glycol and the polyacrylate polymers interlinked via electrostatic interaction (in particular hydrogen bonded), charged surfactant and optionally carbon nanoparticle. The method of fabricating the resin is flexible which allows the selection of charge for the final resin via use of different surfactant and in-situ addition of carbon nanoparticles, without the need of post-addition. In addition, water assisted precipitation method allows energy saving from the sonication method and finetunes the porosity of the resulted resins. The composite was further built for immobilization of allulose isomerase (DM1-514) for converting fructose into allulose. A surprising synergistic catalytic effect was observed for the enzymes immobilized on positively charged acrylic resin composite, and further when carbon nanoparticles are incorporated.

[0052] Accordingly, the present disclosure provides a polymer composite, comprising a matrix formed from polyacrylate, polyethylene glycol (PEG) and ionisable surfactant;

[0053] wherein the polyacrylate at about 9 wt% to about 20 wt% relative to the polymer composite;

[0054] wherein the polyethylene glycol is electrostatically bonded to the polyacrylate, and at about 70 wt% to about 90 wt% relative to the polymer composite; andwherein the ionisable surfactant is homogenously dispersed within the matrix, and at about 1 wt% to about 10 wt% relative to the polymer composite.

[0055] It was found that the inclusion of surfactants may create an electrostatic attraction with the target enzymes to be immobilized, providing further stabilization to the enzyme. In particular, the polyethylene glycol may be hydrogen bonded to the polyacrylate. The ionised ionisable surfactant further increases the electrostatic attraction with the polyethylene glycol and the polyacrylate.

[0056] In some embodiments, the polyacrylate is selected from poly(methacrylate), polymethyl methacrylate, polyethyl methacrylate, poly(2-hydroxylethyl methacrylate). In some embodiments, the polyacrylate is polymethyl methacrylate (PMMA).

[0057] In some embodiments, the polyacrylate is characterised by a molecular weight of about 10,000 g / mol to about 1,000,000 g / mol. In other embodiments, the molecular weight of about 50,000 g / mol to about 1,000,000 g / mol, about 100,000 g / mol to about 1,000,000 g / mol, about 200,000 g / mol to about 1,000,000 g / mol, about 300,000 g / mol to about 1,000,000, about 400,000 g / mol to about 1,000,000 g / mol, about 500,000 g / mol to about 1,000,000 g / mol, about 600,000 g / mol to about 1,000,000 g / mol, or about 700,000 g / mol to about 1,000,000 g / mol. In other embodiments, the molecular weight of about 996,000 g / mol.

[0058] In some embodiments, the polyacrylate at about 10 wt% to about 20 wt% relative to the polymer composite, about 10 wt% to about 18 wt%, about 10 wt% to about 16 wt%, or about 12 wt% to about 16 wt%.

[0059] In some embodiments, PEG is characterised by a molecular weight of about 100 g / mol to about 1000 g / mol. In other embodiments, the molecular weight is about 100 g / mol to about 800 g / mol, about 100 g / mol to about 600 g / mol, or about 200 g / mol to about 600 g / mol. In other embodiments, the molecular weight is about 400 g / mol.It was found that PEG serves as the linkers for the PMMA backbone, which works via hydrogen bonding. A shorter PEG MW serves better as the linker, like rungs on a ladder. By providing a relatively larger amount of PEG at short MW to PMMA, a dense cross-linked network may be formed. While a PEG with small molecular weight is liquid at room temperature and is non-crystalline, after forming a composite, the composite becomes ordered / crystalline with clearly observed DSC peaks.

[0060] In some embodiments, a molecular weight ratio of polyacrylate to PEG is about 1000 to about 3000. In other embodiments, the molecular weight ratio is about 1200 to about 3000, about 1400 to about 3000, about 1600 to about 3000, about 1800 to about 3000, about 2000 to about 3000, about 2200 to about 3000, or about 2400 to about 3000. It was found that a low molecular weight for PEG (as a liquid) allows for better mixing and a greater number of hydrogen bonding.

[0061] In some embodiments, a weight ratio of polyacrylate to PEG is about 1:10 to about 4:10. In other embodiments, the weight ratio is about 1:10 to about 3:10, or about 1:10 to about 2:10. In other embodiments, the weight ratio is 1.8:10.

[0062] As the PEG is of relatively shorter length than the polyacrylate but added at a higher concentration, the amount of hydrogen bonding between the terminal ends of the PEG and oxy moiety of polyacrylate is increased.

[0063] In some embodiments, the ionisable surfactant is ionisable to a positive or negative charge. In this regard, the ionisable surfactant may be ionised surfactant. In some embodiments, the ionisable surfactant is selected from tetraalkylammonium salt or long chain fatty acid. The tetraalkylammonium salt may have an alkyl chain length of C3 to C10. The fatty acid may have an alkyl chain length of Cs to C20. In some embodiments, the ionisable surfactant is selected from stearic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid and tetrabutylammonium salt, tetrapropylammonium salt,alkylbenzyldimethylammonium salt, distearyldimethylammonium salt, cetrimonium salt and behentrimonium salt. The salt may have an anion such as halogen or bis-sulfate.

[0064] In some embodiments, the ionisable surfactant is characterized by an alkyl chain length of C1-C20, or Ci-Cis, or Ci-Cie, or C1-C14, or C1-C12, or C1-C10, or Ci-Cs, or C1-C5.

[0065] In some embodiments, a weight ratio of polyacrylate to ionisable surfactant is about 1:1 to about 5:1. In other embodiments, the weight ratio is about 1:1 to about 4:1, or about 1:1 to about 3:1. In other embodiments, the weight ratio is about 3:1.

[0066] In some embodiments, a weight ratio of polyacrylate to PEG to ionisable surfactant is 15:80:5.

[0067] In some embodiments, the polymer composite further comprising carbon nanoparticles.

[0068] Carbon nanoparticles (zero dimensional) have large surface area and good surface electron conductivity. It is believed that the electron transport on nanoparticles surface may trigger and / or enhance enzyme-like properties. When carbon nanoparticles are put together with enzymes, it was found that a synergistic catalytic effect between nanoparticles and enzyme occurred.

[0069] In some embodiments, the carbon nanoparticles are surface functionalised with PEG.

[0070] In some embodiments, the carbon nanoparticle is about 0.01 wt% to about 1 wt% relative to the polymer composite. In other embodiments, the wt% is about 0.01 wt% to about 0.9 wt%, about 0.01 wt% to about 0.8 wt%, about 0.01 wt% to about 0.7 wt%, about 0.01 wt% to about 0.6 wt%, about 0.01 wt% to about 0.5 wt%, about 0.01 wt% to about 0.4 wt%, about 0.01 wt% to about 0.3 wt%, about 0.01 wt% to about 0.2 wt%, or about 0.01 wt% to about 0.1wt%. In other embodiments, the wt% is about 0.05 wt% to about 0.1 wt%.

[0071] In some embodiments, the carbon nanoparticles are characterized by a particle size of about 1 nm to about 50 nm. In other embodiments, the particle size is about 1 nm to about 40 nm, about 1 nm to about 30 nm, about 1 nm to about 20 nm, about 1 nm to about 10 nm, or about 1 nm to about 5 nm.

[0072] In some embodiments, the polymer composite is an porous polymer framework. The porous polymer framework may be characterised by a stochastic or random distribution of pores. As shown in the SEM images, after formation of the pores, they are not fused together but retain their individual discrete pore structure. The pores may be characterised by a pore size distribution. Such porous polymer framework has at least one endothermic peak when measured using DSC. In this regard, the polymer composite may have some degree of crystallinity.

[0073] In some embodiments, the polymer composite is characterised by a particle size of about 100 pm to about 600 pm. In other embodiments, the particle size is about 200 pm to about 600 pm, about 300 pm to about 600 pm, or about 300 pm to about 500 pm.

[0074] In some embodiments, the polymer composite is characterized by a endothermic peak temperature. In some embodiments, the polymer composite is characterized by a first endothermic peak temperature of about 65 °C to about 85 °C. In other embodiments, the first endothermic peak temperature of about 65 °C to about 80 °C, about 65 °C to about 75 °C, or about 65 °C to about 70 °C.

[0075] In some embodiments, the polymer composite is characterized by a second endothermic peak temperature of about 90 °C to about 115 °C. In some embodiments, the polymer composite is characterized by a second endothermic peak temperature of about 95 °C to about 115 °C, about 100 °C to about 115 °C, about 105 °C to about 115 °C, or about 110 °C to about 115 °C.

[0076] In some embodiments, the polymer composite is characterised by a degree ofcrystallinity of about 1% to about 20%. In other embodiments, the polymer composite is characterised by a degree of crystallinity of about 1% to about 18%, about 1% to about 16%, about 1% to about 14%, about 1% to about 12%, or about 4% to about 12%. In other embodiments, the degree of crystallinity of about 4.4%. In another embodiment, the degree of crystallinity of about 19%.

[0077] In some embodiments, the polymer composite is characterized by a porous structure. The polymer composite may be nanoporous. The polymer composite may be microporous, mesoporous and / or macroporous. As defined in the International Union of Pure and Applied Chemistry (IUPAC), porous materials are classified into three categories based on different pore sizes: microporous (<2 nm), mesoporous (2-50 nm) or macroporous (>50 nm) materials.

[0078] The polymer composite as formed comprises pores of a random nature. In other words, the porous structure is not hierarchical.

[0079] In some embodiments, the polymer composite is characterized by a pore size distribution of about 8 nm to about 250 nm. In this regard, the polymer composite may comprise a combination of micropores, mesopores and macropores. In some embodiments, the polymer composite is characterized by a pore size distribution of about 8 nm to about 30 nm, about 8 nm to about 28 nm, about 8 nm to about 26 nm, about 8 nm to about 24 nm, about 8 nm to about 22 nm, or about 8 nm to about 20 nm. In some embodiments, the polymer composite is characterized by a pore size distribution of about 10 nm to about 250 nm, about 20 nm to about 250 nm, about 30 nm to about 250 nm, about 40 nm to about 250 nm, about 50 nm to about 250 nm, about 60 nm to about 250 nm, about 70 nm to about 250 nm, about 80 nm to about 250 nm, about 90 nm to about 250 nm, or about 100 nm to about 250 nm. In some embodiments, the polymer composite is at least mesoporous.

[0080] In some embodiments, the polymer composite is characterized by a pore size of more than about 8 nm.In some embodiments, the pores having a pore size distribution of about 30 nm to about 250 nm is more than about 50% relative to the total pores. In other embodiments, the pores are more than about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, or about 90% relative to the total pores.

[0081] In some embodiments, the polymer composite is characterized by a Vpore / V-total of more than about 80%, about 82%, about 84%, about 86%, about 88%, or about 90%. In some embodiments, the polymer composite is characterized by a Vpore / V-total of about 80% to 100%. The term Vpore / V-total refers to the ratio of the volume of mesopores and / or macropores to the total pore volume of the polymer composite. The total pore volume encompasses micropores, mesopores, macropores, or any combination thereof. A higher Vpore / V-total value indicates a greater proportion of larger pores within the polymer composite, which may be useful for mass transport and diffusion in enzyme adsorption. Vpore may be determined using the Barrett-Joyner-Halenda (BJH) method, while V-total may be obtained using the Brunauer-Emmett-Teller (BET) method.

[0082] In some embodiments, the polymer composite is characterized by a BET surface area of about 0.6 m2 / g to about 11 m2 / g. In some embodiments, the polymer composite is characterized by a BET surface area of about 1 m2 / g to about 11 m2 / g, about 2 m2 / g to about 11 m2 / g, about 3 m2 / g to about 11 m2 / g, about 4 m2 / g to about 11 m2 / g, or about 5 m2 / g to about 11 m2 / g.

[0083] In some embodiments, the polymer composite is characterized by a pore volume is about 0.02 cm3 / g to about 0.4 cm3 / g. In some embodiments, the polymer composite is characterized by a pore volume is about 0.03 cm3 / g to about 0.4 cm3 / g, about 0.05 cm3 / g to about 0.4 cm3 / g, about 0.1 cm3 / g to about 0.4 cm3 / g, or about 0.2 cm3 / g to about 0.4 cm3 / g.

[0084] The present disclosure also provides an immobilised enzyme, wherein the enzyme is immobilised on a polymer composite as disclosed herein.

[0085] In some embodiments, the enzyme is adsorbed on the polymer composite. Theenzyme may be physically adsorbed on the surface and / or entrapped in the pores of the polymer composite. The enzyme may be electrostatically adsorbed on the surface and / or pores of the polymer composite. This may be resulting from the interaction of the enzyme with the ionisable surfactant, PEG and the optional carbon nanoparticles. The electrostatic force may be hydrogen bonding.

[0086] In general, the interaction between the enzyme, depending on its isoelectric point, and the composite may be influenced by their respective surface charges at specified pH. An enzyme exhibiting a net positive charge may demonstrate enhanced adsorption when associated with a negatively charged polymer composite at specified pH. Conversely, an enzyme exhibiting a net negative charge may exhibit enhanced adsorption when associated with a positively charged polymer composite at specified pH. This electrostatic complementarity may facilitate stable immobilization and optimize catalytic efficiency. For example, allulose isomerase has isoelectric point of about pH 4.5 to pH 6, thus at pH 6, the enzyme is slightly acidic and adsorbs well on a positively charged polymer composite.

[0087] In certain embodiments, the polymer composite is characterised by an enzyme loading capacity of at least 30 mg / g of the composite. In some embodiments, the polymer composite is characterised by an enzyme loading capacity of about 20 mg / g of polymer composite to about 70 mg / g of polymer composite. In some embodiments, the enzyme loading capacity is about 20 mg / g to about 60 mg / g, about 20 mg / g to about 50 mg / g, or about 20 mg / g to about 40 mg / g.

[0088] In some embodiments, the enzyme is selected from allulose isomerase, lipase, esterase, cutinase, PETase, or a combination thereof.

[0089] In some embodiments, the immobilised enzyme is characterised by an enzyme activity of at least 6 times relative to a free enzyme. In some embodiments, the immobilised enzyme is characterised by an enzyme activity of at least 7, 8, 9, 10, 15, or 20 times relative to a free enzyme.

[0090] In some embodiments, when the polymer composite comprises positivelyionisable surfactant and carbon nanoparticles, the immobilised enzyme is characterised by an enzyme activity of at least 5 times relative to an enzyme immobilised on a polymer composite without carbon nanoparticles. In other embodiments, the enzyme activity is at least 6, 7 or 8 times relative to an enzyme immobilised on a polymer composite without carbon nanoparticles.

[0091] The present disclosure also provides a method of fabricating a polymer composite, comprising:

[0092] a) dissolving polyacrylate, polyethylene glycol and ionisable surfactant in a solvent to form a solution; and

[0093] b) mixing the solution with a non-solvent in order to precipitate the polyacrylate, polyethylene glycol and ionisable surfactant to for the polymer composite.

[0094] It was found that using a non-solvent, a "softness" is introduced into the matrix in that the association or crosslinking between polyacrylate and PEG is not excessive. This is in contrast to sonication, which imparts a "hardness" into the matrix, forming pores of relatively smaller sizes and having less total pore volume of pores at P / Po=0.99.

[0095] The solution may be formed in a polar solvent. The solution may be formed in an aprotic solvent. The solvent may be acetone.

[0096] The non-solvent is a substance that is incapable of dissolving a component of a solution. For example, the polyacrylate may be hydrophobic. Accordingly, an aqueous solution may be used to cause the polyacrylate to precipitate out, the process of which causes PEG and ionisable surfactant to associate and form the polymer composite.

[0097] The term 'aqueous medium' or 'aqueous solution' used herein refers to a water based solvent or solvent system, and which comprises of mainly water. Such solvents can be either polar or non-polar, and / or either protic or aprotic. Solvent systems refer to combinations of solvents which resulting in a final single phase. Both ’solvents' and 'solvent systems' can include, and is not limited to, pentane,cyclopentane, hexane, cyclohexane, benzene, toluene, dioxane, chloroform, diethylether, dichloromethane, tetra hydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, nitromethane, propylene carbonate, formic acid, butanol, isopropanol, propanol, ethanol, methanol, acetic acid, ethylene glycol, diethylene glycol or water. Water based solvent or solvent systems can also include dissolved ions, salts and molecules such as amino acids, proteins, sugars and phospholipids. Such salts may be, but not limited to, sodium chloride, potassium chloride, ammonium acetate, magnesium acetate, magnesium chloride, magnesium sulfate, potassium acetate, potassium chloride, sodium acetate, sodium citrate, zinc chloride, HEPES sodium, calcium chloride, ferric nitrate, sodium bicarbonate, potassium phosphate and sodium phosphate.

[0098] In this regard, the aqueous solution may comprise a salt, acid or base for ionising the ionisable surfactant.

[0099] In some embodiments, step a) further comprises adding carbon nanoparticles to the solution.

[0100] In some embodiments, the method further comprises a step of purifying the polymer composite. For example, the polymer composite may be dialysed, or filtered.

[0101] Examples

[0102] The invention will be disclosed in more details below with examples hereafter. Nevertheless, these examples are not to be treated as limiting the scope of the current invention.

[0103] Preparation of resins

[0104] In one embodiment, polymethyl methacrylate (PMMA, MW = 996k by GPC, sigma-aldrich) was selected as the representative polyacrylate. Polymethyl methacrylate powder are crystals with high rigidity, lack of porosity and therefore are not suitable for hosting enzymes. To achieve porosity, polyethylene glycol was used to create heterogeneous structure, aspolyethylene glycol could form hydrogen bonding with PMMA (Figure 1). To further introduce anionic functional groups, stearic acid (C17-COOH) was also blended into the composite. To prepare the composite, all three components are dissolved in acetone. To achieve the solid composite, the liquid is subjected to sonication, where precipitation of the composite is formed. The precipitation appears to be white solid with very high hardness.

[0105] In a parallel embodiment, carbon nanoparticles are incorporated into the composite. Carbon nanoparticles have been reported to show catalytic effect, and PMMA composite containing carbon nanoparticles might serve as better host for enzyme with potential synergy in catalytic effect compared to the counterpart without carbon nanoparticles.

[0106] In a further embodiment, water was introduced as assist for anti-solvent process to obtain precipitate of composite. As compared the sonication assisted precipitation, the resulted composite solid appear soft, increasing its processibility for further treatment, e.g., granulation.

[0107] In other embodiments, cationic groups are introduced to the composite via blending with a quaternary ammonium compound (i.e., tetrabutylammonium bis-sulfate). The quat based composite was generated with water assisted process, since the high hardness of the composite from sonication process is less desirable. The cationic composite with carbon nanoparticles were also prepared accordingly.

[0108] Example 1 (Ex. 1): PMMA (MW=996k, Sigma-Aldrich), polyethylene glycol (MW=400, Sigma-Aldrich), and stearic acid (Sigma-Aldrich) were charged to acetone in the ratio of 15:80:5 by weight and dissolved completely. The organic mixture was then sonicated to form white precipitate, which was further collected by suction filtration and vacuum dried.

[0109] Example la (Ex. la): Preparation of PEG containing carbon nanoparticles. 6 parts of citric acid & 3 parts of glycerol are added to 90 parts of PEG (MW=400, Sigma-Aldrich) and stirred at room temperature until all dissolved. The mixturewas then carbonized at 210 °C for 3 hours. Brown colour liquid was obtained, which is PEG containing carbon nanoparticles (about 2 nm to about 5 nm).

[0110] Example 2 (Ex. 2): PMMA (MW=996k, Sigma-Aldrich), polyethylene glycol containing carbon nanoparticles (Ex. la), and stearic acid (Sigma-Aldrich) were charged to acetone in the ratio of 15:80:5 by weight and dissolved completely. The organic mixture was then sonicated to form off-white precipitate, which was further collected by suction filtration and vacuum dried.

[0111] Example 3 (Ex. 3): PMMA (MW=996k, Sigma-Aldrich), polyethylene glycol (MW=400, Sigma-Aldrich), and stearic acid (Sigma-Aldrich) were charged to acetone in the ratio of 15:80:5 by weight and dissolved completely. Water was then added into the organic mixture to form white precipitate, which was further collected by suction filtration and vacuum dried.

[0112] Example 4 (Ex. 4): PMMA (MW=996k, Sigma-Aldrich), polyethylene glycol containing carbon nanoparticles (Ex. la), and stearic acid (Sigma-Aldrich) were charged to acetone in the ratio of 15:80:5 by weight and dissolved completely. Water was then added into the organic mixture to form off-white precipitate, which was further collected by suction filtration and vacuum dried.

[0113] Example 5 (Ex. 5): PMMA (MW=996k, Sigma-Aldrich), polyethylene glycol (MW=400, Sigma-Aldrich), and tetrabutylammonium bis-sulfate (Sigma-Aldrich) were charged to acetone in the ratio of 15:80:5 by weight and dissolved completely. Water was then added into the organic mixture to form white precipitate, which was further collected by suction filtration and vacuum dried.

[0114] Example 6 (Ex. 6): PMMA (MW=996k, Sigma-Aldrich), polyethylene glycol (MW=400, Sigma-Aldrich), and tetrabutylammonium bis-sulfate (Sigma-Aldrich) were charged to acetone in the ratio of 15:80:5 by weight and dissolved completely. Then carbon nanoparticles (~50 nm, Sigma-Aldrich) was added to the mixture and further mixed. Water was then added into the organic mixture to form light-black precipitate, which was further collected by suction filtration and vacuum dried.Characterization of resins

[0115] The 6 composites have been measured via DSC to understand their noncrystalline properties. Table 1 summarizes the DSC peaks for resin example 1-6. For all 4 resins composite prepared from PMMA + PEG + stearic acid, single sharp peaks were observed. The endothermic peaks for Ex. 1 - 4 are observed around 67 to 72 °C. The 2 resins prepared from PMMA + PEG + TBABiS (i.e.. Ex. 5 & 6) have two endothermic peaks, respectively, which indicate two types of crystalline structure co-exist in the composite. Nonetheless, the peaks observed confirmed the composite are uniformly constructed. The process method disclosed thus is an effective method to generate homogenous acrylate-based resins. It is worth noting that pure PMMA usually has a DSC peak at very high temperature, which is due to its decomposition (e.g., > 350 °C).

[0116] Table 1 Summary of DSC peaks for Ex. 1 to Ex. 6

[0117]

[0118] These resins are also confirmed to be porous making them potentially to be an efficacious enzyme host. The SEM images of typical acrylate stearic acid resins and acrylate quat resins are shown in Figure 2. The resins possess irregular pores with interconnected network surface. Similar surface structures are also observed for other reported acrylate polymeric composites. The rough surface symbolizes the potential use of these resins for enzyme adsorption.

[0119] A BET (Brunauer, Emmett and Teller) test was further conducted to measure the specific surface area, pore size distribution, and total pore volume. Table 2 summarize the BET results for Ex. 1 to Ex. 6. The pore volume in reported in Table 2 is the Single point adsorption Total pore volume (V-total) of pores at P / Po= 0.99. The average pore size reported in Table 2 refers to the Adsorption average pore width (4V / A by BET). BJH pore size distribution was analyzed ineach test, such that, the BJH Adsorption cumulative volume of pores between 1.700 nm and 300.000 nm width and BJH Desorption cumulative volume of pores between 1.700 nm and 300.000 nm width were measured.

[0120] The resins prepared from non-water process (i.e., Ex. 1 & 2) provided average pore size of ~17 nm and ~9 nm, respectively, indicating that most of the pores are nanopores. When the precipitation process was performed with water, the obtained resins Ex. 3 has a much bigger average pore size of ~180 nm. An optimization process with water turns most of the pores within the resin from nanoporous to at least mesoporous. The sample with carbon nanoparticles (Ex.

[0121] 4) prepared with water possesses average pore size of ~32 nm, which is smaller compared to the counterpart with no carbon nanoparticles (Ex. 3). This could be due to that carbon nanoparticles sit in the pores and alter the BET size measurement. The resins prepared with quat with water process also have relatively big average pore size of ~150 nm and the counterpart with carbon nanoparticles showed similar trend of reduced average pore size of ~15 nm.

[0122] Table 2 Summary of BET results for Ex. 1 to 6.

[0123]

[0124] Results on enzyme immobilization

[0125] Enzyme loading onto the resins prepared from sonication method and water method are compared. Isomerase DM1-514 (in-house) purified protein is enzyme converting fructose into allulose, which was used as a representative enzyme. Figure 3 showed the loading of DM1-514 on to Ex. 1 and Ex. 2. Columns 1 and 3 are the results from working up the resin (bounded enzyme), while columns 2 & 4 are from the supernatant after enzyme loading (non-bounded enzyme). The loading of enzyme on the resin appears to be very low. Ex. 1 and2 are prepared from sonication assisted method, and the resins are hard in nature with very small (17 nm for Ex. 1 and 9 nm for Ex. 2) average pore sizes. These two resins can be categorized as mainly nanoporous and may not be a carrier for hosting DM1-514. In contrast, the loading of DM1-514 onto Ex. 3 and Ex. 4 are more significant. As shown in Figure 4, columns 1 and 3 are from the supernatant after enzyme loading (non-bounded enzyme) and columns 2 and 4 are from the results from working up the resin (bounded enzyme). Ex. 3 and 4 are mainly mesoporous or microporous materials with average pore sizes of 180 nm for Ex. 3 and 31 nm for Ex. 4. Such results confirmed that the PMMA resins in mesoporous nature prepared from water assisted method are more suitable for loading of enzymes (i.e., DM1-514).

[0126] The activity of the immobilized DM1-514 enzymes is evaluated and compared. It is worth noting that non-immobilized DM1-514 enzyme are susceptible to heat, where the activity is almost completely lost after 0.5 h of heating at 70 °C. In addition, enzymes without immobilization lack of the ability to be used in cycles and have to be separated after the reaction.

[0127] Figure 5 shows the activity data (expressed in allulose conversion %) at Ih under 60 °C. The flow rate was kept the same at 0.5 miymin of 100 g / L fructose containing 0.10 mM C0CI2 for all immobilized enzyme samples. The activity for both Ex. 3-i and Ex. 4-i are similar but not high (<10%). The activity between Ex. 5-i and Ex. 6-i are very contrary, with 2.7% for Ex. 5-i and 18.9% for Ex.

[0128] 6-i, which is 7 times higher. With the charge and carbon nanoparticles taken into consideration, the fact that resins from Ex. 6 performed best as the immobilization host for DM1-514 may be ascribed to the cationic nature and synergistic effect from carbon nanoparticles.

[0129] Discussion

[0130] The process of making the composite with sonication is less desirable compared to the process using water. Firstly, the use of sonication consumes further energy / electricity and is more complicated than use water for anti-solvent precipitation. Secondly, the resulted resin from sonication is very hard and therefore not easy for further treatment. The use of water removes the weaklyassociated PEG in the composite, only the properly hydrogen bonded PEG remained within the polymer composite after water addition. The water assisted process yields composite with single DSC peak in Ex. 3 and Ex. 4 indicates that the process is able to produce homogeneous composite structure. The water assisted process yields composite with dual DSC peaks in Ex. 5 and Ex. 6 are still macroscopically uniform as evidenced by SEM images.

[0131] With the backbone of PMMA & PEG, the process for making the composite is flexible, which allows further adjusting the charge and in-situ addition of nanoparticles. In specific, with a negative surfactant (e.g., stearic acid) or a positive surfactant (e.g., quat), negatively charged and positively charged composite can be prepared, respectively. Further addition of nanoparticles (i.e., carbon nanoparticles) could also be easily achieved in situ with the formation of composite backbones.

[0132] The composition and the catalytic effect would not be known with prior knowledge and maybe varied for different enzymatic reaction. Synergistic from carbon dots and allulose isomerase (i.e., DM1-514) was observed for catalytic reaction generating allulose from fructose. In the demonstrated catalytic reactions of fructose to allulose using immobilized DM1-514, the enzymes immobilized on negatively charged acrylic composite generated on par and low activity with or without the carbon nanoparticles. In the identical experimental setup, the enzymes immobilized on positively charged acrylic resin composite with carbon nanoparticles give a 7 times higher activity than the counterparts without carbon nanoparticles. The achieved results are surprising, as the synergistic effect only happened on the enzymes immobilized on positively charged resin composite.

[0133] The technology is thus sustainable using low energy process and no heating, flexible on charge selection, and highly efficacious showing a synergistic effect. It can be applied widely to build immobilized enzyme and use for specific catalytic reactions with respect to the enzyme. These include various large-scale industries, e.g., food, detergent, textile, pharmaceutical and recycling, etc .It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

[0134] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0135] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0136] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

Claims1. A polymer composite, comprising a matrix formed from polyacrylate, polyethylene glycol (PEG) and ionisable surfactant;wherein the polyacrylate at about 9 wt% to about 20 wt% relative to the polymer composite;wherein the polyethylene glycol is electrostatically bonded to the polyacrylate, and at about 70 wt% to about 90 wt% relative to the polymer composite; and wherein the ionisable surfactant is homogenously dispersed within the matrix, and at about 1 wt% to about 10 wt% relative to the polymer composite.

2. The polymer composite according to claim 1, wherein the polyacrylate is selected from poly(methacrylate), polymethyl methacrylate, polyethyl methacrylate, poly(2-hydroxylethyl methacrylate).

3. The polymer composite according to claim 1 or 2, wherein the polyacrylate is characterised by a molecular weight of about 10,000 g / mol to about 1,000,000 g / mol.

4. The polymer composite according to any one of claims 1 to 3, wherein PEG is characterised by a molecular weight of about 100 g / mol to about 1000 g / mol.

5. The polymer composite according to any one of claims 1 to 4, wherein a molecular weight ratio of polyacrylate to PEG is about 1000 to about 3000.

6. The polymer composite according to any one of claims 1 to 5, wherein a weight ratio of polyacrylate to PEG is about 1:10 to about 4:10.

7. The polymer composite according to any one of claims 1 to 6, wherein the ionisable surfactant is ionised to a positive or negative charge.

8. The polymer composite according to any one of claims 1 to 7, wherein the ionisable surfactant is selected from tetraalkylammonium salt and long chainfatty acid or salt thereof.

9. The polymer composite according to any one of claims 1 to 8, wherein the ionisable surfactant is selected from stearic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid and tetrabutylammonium salt, tetrapropylammonium salt, alkylbenzyldimethylammonium salt, distearyldimethylammonium salt, cetrimonium salt and behentrimonium salt.

10. The polymer composite according to any one of claims 1 to 9, wherein a weight ratio of polyacrylate to ionisable surfactant is about 1:1 to about 5:1.

11. The polymer composite according to any one of claims 1 to 10, wherein a weight ratio of polyacrylate to PEG to ionisable surfactant is 15:80:5.

12. The polymer composite according to any one of claims 1 to 11, wherein the polymer composite further comprises carbon nanoparticles.

13. The polymer composite according to claim 12, wherein the carbon nanoparticle is about 0.01 wt% to about 1 wt% relative to the polymer composite.

14. The polymer composite according to any one of claims 1 to 13, wherein the polymer composite is a porous solid characterised by a particle size of about 100 pm to about 600 pm.

15. The polymer composite according to any one of claims 1 to 14, wherein the polymer composite is characterised by a degree of crystallinity of about 1% to about 20%.

16. The polymer composite according to any one of claims 1 to 15, wherein the polymer composite is characterized by a first endothermic peak temperature of about 65 °C to about 85 °C, and optionally a second endothermic peak temperature of about 90 °C to about 115 °C.

17. The polymer composite according to any one of claims 1 to 16, wherein the polymer composite is characterized by a pore size of about 8 nm to about 250 nm.

18. The polymer composite according to any one of claims 1 to 17, wherein the polymer composite is characterized by a Vpore / V-total of more than about 80%.

19. An immobilised enzyme, wherein the enzyme is immobilised on a polymer composite according to any one of claims 1 to 18.

20. The immobilised enzyme according to claim 19, wherein the enzyme is adsorbed on the polymer composite.

21. The immobilised enzyme according to claim 19 or 20, wherein the enzyme is selected from allulose isomerase, lipase, esterase, cutinase, PETase, or a combination thereof.

22. The immobilised enzyme according to any one of claims 19 to 21, wherein the immobilised enzyme is characterised by an enzyme activity of at least 6 times relative to a free enzyme.

23. The immobilised enzyme according to any one of claims 19 to 22, wherein when the polymer composite comprises positively ionisable surfactant and carbon nanoparticles, the immobilised enzyme is characterised by an enzyme activity of at least 5 times relative to an enzyme immobilised on a polymer composite without carbon nanoparticles.

24. A method of fabricating a polymer composite, comprising:a) dissolving polyacrylate, polyethylene glycol and ionisable surfactant in a solvent to form a solution; andb) mixing the solution with a non-solvent in order to precipitate the polyacrylate, polyethylene glycol and ionisable surfactant to form the polymercomposite.

25. The method according to claim 24, wherein step a) further comprises adding carbon nanoparticles to the solution.

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